Methods for testing anti-thrombotic agents

ABSTRACT

The invention provides a transgenic non-human animal expressing von Willebrand Factor A1 protein containing at least one mutation selected from the group consisting of: 1263P&gt;S, 1269N&gt;D, 1274K&gt;R, 1287M&gt;R, 1302G&gt;D, 1308H&gt;R, 1313R&gt;W, 1314I&gt;V, 1326R&gt;H, 1329L&gt;I, 1330E&gt;G, 1333A&gt;D, 1344T&gt;A, 1347I&gt;V, 1350T&gt;A, 1370G&gt;S, 1379H&gt;R, 1381T&gt;A, 1385T&gt;M 1391P&gt;Q, 1394A&gt;S, 1397L&gt;F, 1421S&gt;N, 1439L&gt;V, 1442G&gt;S, 1449R&gt;Q, 1466A&gt;P, 1469Q&gt;L, 1472Q&gt;H, 1473V&gt;M, 1475H&gt;Q, 1479S&gt;G, and any combination thereof.

This application is a continuation-in-part of International Application No. PCT/US2007/015043 filed on Jun. 28, 2007, which claims the benefit of priority of U.S. Ser. No. 60/817,600 filed on Jun. 29, 2006, the contents of which are hereby incorporated in their entirety.

GOVERNMENT INTERESTS

This invention was made with support from the U.S. Federal Government under Grant No. 5RO1HL63244-7 awarded by the National Heart, Lung, and Blood Institute (NHLBI). As such, the United States government has certain rights in this invention.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

BACKGROUND OF THE INVENTION

The ability of platelets to rapidly stick to the damaged wall of arterial blood vessels is critical for preventing blood loss (hemorrhage). Inappropriate deposition of these hemostatic cells in arterial blood vessels due to pathological disease processes such as atherosclerosis can result in lack of blood flow to vital organs such as the heart and brain. Thus a delicate balance exists between providing adequate hemostasis without causing blockage of blood vessels by excessive platelet deposition (a.k.a. thrombus formation).

von Willebrand Factor (VWF) is a multidomain, plasma glycoprotein of complex multimeric structure which is synthesized by vascular endothelial cells and megakaryocytes (1-3) (FIG. 1A). Its presence in the blood is vital to maintaining the integrity of the vasculature. To accomplish this task, VWF forms a “bridge” between the injured vessel wall and platelets by virtue of its ability to interact with extracellular matrix components, such as collagen, and receptors expressed on platelets, such as glycoprotein Ib alpha (4-9). It also binds to and confers stability to factor VIII (10). The importance of this glycoprotein in hemostasis is underscored by the occurrence of clinical bleeding when the plasma VWF levels fall below 50 IU/dL (type I von Willebrand's disease, VWD), or when functional defects in the protein occur (type 2 VWD) (11,12).

Upon surface immobilization of VWF at sites of vascular injury, it is the role of the A1 domain of VWF (residues 1260-1480) to initiate the process of platelet deposition at sites of vascular injury and under conditions of high rates of shear flow (>1,000 s⁻¹; Ruggeri, Z. M. et al. Blood. 108, 1903-1910 (2006)). The critical nature of this interaction is exemplified by the bleeding disorder, termed type 2M VWD, which results from the incorporation of loss-of-function mutations within this domain that perturb interactions with GPIb alpha (Sadler, J. E. et al. (2006) J. Thromb. Haemost. 4, 2103-2114; Rabinowitz, I. et al. (1992) Proc. Natl. Acad. Sci. USA 89, 9846-9849; Cruz et al., (2000) J. Biol. Chem. 275, 19098-19105). In addition, recombinant VWF multimers lacking the A1 domain cannot support platelet adhesion at high rates of flow despite retaining the ability to interact with collagen (Sixma et al., (1991) Eur J Biochem. 196:369-75).

The structure of the A1 domain includes the α/β fold with a central β-sheet flanked by α-helices on each side as well as one intra-disulfide bond (Cys1272-Cys 1458), but no MIDAS motif (Emsley et al., (1998)J Biol Chem. 273:10396-401). Its overall shape is cuboid, with the top and bottom faces forming the major and minor binding sites, respectively, that interact with the concave surface of GPIbα. The most extensive contact site buries ˜1700 Å² of surface area, interacting with LRR five to eight and the C-terminal flank of the GPIbα (Huizinga, E. G. et al. (2002) Science 297, 1176-1179). For this to occur, the β-switch region of this platelet receptor undergoes a conformation change so that it aligns itself with the central beta sheet of the A1 domain. The smaller site (˜900 Å²) accommodates the binding of the β-finger and the first LRR of GPIbα, an event that appears to require the displacement of the amino-terminal extension of the A1 domain. Based on these findings as well as the preferential localization of mutations in humans within this region, which enhance GPIbα binding, it is speculated that the amino-terminal extension regulates the adhesive properties of this domain. This is also supported by the fact that recombinant A1 proteins lacking this extension have a higher affinity for this platelet receptor (Sugimoto et al., (1993) J Biol Chem. 268:12185-92). Despite these observations, the physiological relevance of such structural changes in this receptor-ligand pair remains to be determined as well as the contribution of other domains to this process.

In addition to its role in hemostasis, VWF also contributes to pathological thrombus formation on the arterial side of the circulation. This may be the consequence of injury to the blood vessel wall from inflammatory disease states and/or medical/surgical interventions. Pathological thrombus formation is the leading cause of death in the Western world. Thus, pharmaceutical companies have committed considerable resources towards the research and design of drugs to prevent or treat thrombosis. However, there remains an urgent need to develop new and improved therapies such as those aimed at reducing platelet and/or VWF interactions with the injured arterial wall. One major hurdle hindering drug development in this field is the lack of an appropriate small animal model of thrombosis to test promising therapies. For instance, differences in the structure or isoform of protein receptors or ligands on mouse vs. human platelets that are critical for the activation and/or binding of these cells to the injured vessel wall preclude testing of drugs developed against human platelets in a mouse model of thrombosis. Moreover, this issue cannot be overcome by simply transfusing mice with human platelets as we have observed that mouse VWF does not support significant interactions with human cells (see below). Thus, the development of a “humanized” mouse model of hemostasis and thrombosis would potentially expedite drug discovery and testing.

That said, we have discovered that only one amino acid difference between mouse and human VWF-A1 domains accounts for most of the inability of the former to interact with human platelets and vice versa. With this knowledge in hand, we have genetically altered a mouse to express VWF that contains this amino acid found in human VWF-A1, imparting on it the ability to support adhesion of human platelets to a level observed for its human counterpart. As a result, not only are we uniquely poised to better understand the molecular mechanisms governing human platelet binding at sites of vascular injury in vivo, but now have the capability to perform pre-clinical testing of anti-thrombotic agents and targeted molecular imaging agents directed against human platelet cells in a living animal. The material contained within this document describes the features of this unique biological platform for drug testing the testing of drugs and targeted molecular imaging agents.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic representation of the prepro form of VWF. From top to bottom: repeated homologous regions; A1 and A3 disulfide loops and functional domains of the mature VWF subunit.

FIG. 1B is an illustration depicting the sequential adhesive and activation events that promote platelet deposition at sites of vascular injury.

FIG. 2 is a model depicting the location of residues in the human VWF-A1 domain that if mutated, diminish GPIb alpha-mediated platelet binding in flow. Type 2M mutations are in red, residues identified by alanine scanning mutagenesis are in blue, and residues identified by crystal structure are in green.

FIG. 3, panel A, is a structure model depicting residues associated with type 2M (red) or type 2B (yellow) VWD. Panel B represents a silver stained gel that clinically depicts a type 2B VWD disease state individual, which is characterized by a loss of circulating high molecular weight VWF multimers (HMWM, FIG. 3B, Lane 2).

FIG. 4 shows structure models of the human VWF-A1 domain. FIG. 4A represents the location of the Ile1309 mutation and its proposed effects on residues critical for GPIb binding. FIG. 4B shows the loss of the isoleucine methyl group allows a water molecule to enter, which ultimately results in changes in orientation of the G1324 peptide plane and the side chain of H1326 as depicted, residues critical for GPIb binding.

FIG. 5 is a space-filling model of the botrocetin-A1 complex with sites involved in GPIb alpha binding and location of type 2B mutations indicated (panel A), wherein botrocetin does not alter the conformation of VWF-A1. In panel B, minor conformational changes in the A1 domain are represented. Uncomplexed (blue) and complexed (green) mutant domains are superimposed onto the WT structure (red).

FIG. 6 is schematic wherein the uncomplexed A1 domain, an amino-terminal extension (pink) appears to block a binding site for the amino-terminal β-hairpin (orange arrows) of GPIb alpha. Binding requires the amino-terminal extension of A1 to move, and also induces the β-switch (yellow loop) of GPIb alpha to form a β-strand motif.

FIG. 7 depicts microscope images wherein the use of platelets in lieu of recombinant proteins or transfected cells as the immobilized substrate enables evaluation of GPIb alpha in its native form (i.e. correct orientation and proper post-translational modification). Platelet coverage of <10% can be bound in this manner and can remain relatively unactivated for up to 30 min as evident by morphology on light microscopic examination (FIG. 7A) and lack of expression of P-selectin by fluorescence microscopy (FIG. 7B).

FIG. 8 represents quantitations of bead-platelet interactions under flow. FIGS. 8A and 8B demonstrates the direct visualization of bead-platelet interaction under flow (60×DIC microscopy). An approaching bead moving at a velocity of 609±97 um/sec (wall shear stress of 1.5 dyn cm⁻²) is captured by a surface-immobilized platelet at (t=12.8 msec), pivots a distance of less than 3 μm in under 40 msec, and is then released after a pause time of t_(p)=228.2 msec into the flow stream (escape velocity=288+/−90.4 μm/sec). FIG. 8C depicts representative experiments of k_(off) values for WT human VWF-A1 coated beads based on a distribution of interaction (pause) times. FIG. 8D shows that the kinetics of the GPIb alpha tether bond are identical whether platelets are metabolically inactivated or fixed in paraformaldehyde to prevent activation upon surface-immobilization.

FIG. 9 represents the deduced single-letter amino acid sequence of mouse VWF-A1 domain (M VWF) compared to its human counterpart (H VWF) from amino acid 1260 to 1480. The locations of cysteines forming the loop structure are numbered (1238 and 1472) and differences in residues are highlighted in red. Conversion of the arginine (R) in the mouse A1 domain to histidine (H) as found in its human counterpart (blue χ) enables mouse VWF to bind human platelets.

FIG. 10 represents graphs of ristocetin-induced platelet aggregation assays (RIPA). Concentrations of the ristocetin modulator known to cause agglutination of human platelets (˜1.0 mg/ml) had no effect using murine platelet rich plasma (FIG. 10B, FIG. 10D). Incubation of murine platelet rich plasma (PRP) with thrombin resulted in >90% platelet aggregation (FIG. 10A). Concentrations of ≧2.5 mg/ml of modulator resulted in murine platelet aggregation (30%, FIG. 10C).

FIG. 11 depicts a multimer gel analysis of purified VWF from human (lane 1, FIG. 11A) and mouse (lane 2, FIG. 11A) plasma. The ability of human and mouse VWF to mediate platelet adhesion in flow was determined in order to evaluate platelet interactions between human and murine VWF with GPIb alpha, as depicted in the bar graph of FIG. 11B. Surface-immobilized murine VWF supports adhesion of syngeneic platelets (1×10⁸/ml) at a shear rate encountered in the arterial circulation (1600 s⁻¹) as observed for the human plasma protein (FIG. 11B, first panel). In contrast, murine VWF did not support significant interactions with human platelets and vice versa.

FIG. 12 is an image of a gel of mouse and human VWF-A1 highly purified protein, which was dialyzed against 25 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.8. SDS-PAGE analysis revealed a prominent protein band of 34,000 Da for mouse VWF-A1 under non-reducing conditions.

FIG. 13 depicts bar graphs of a series of in vitro flow chamber assays performed to assess platelet adhesion, wherein human or murine platelets (5×10⁷/ml) were infused through a parallel plate flow chamber containing glass cover slips coated with either human (H) VWF-A1 or murine (M) VWF-A1 protein (100 μg/ml final concentration) at a shear rate of 800 s⁻¹. M VWF-A1 protein supported platelet adhesion as efficiently as its human counterpart under physiological flow conditions (FIG. 13A). The translocation of mouse platelets occurred to a similar degree as its human counterpart under physiological flow conditions (FIG. 13B). However, human platelets had a reduced capacity to interact with M VWF-A1 protein and mouse platelets had a reduced capacity to interact with H VWF-A1 protein in flow.

FIG. 14, panel A, represents purified bacterial His-tagged VWF-A1 protein and non-His tagged VWF-A1 protein that was analyzed by SDS-PAGE (12.5%) under non-reducing and reducing conditions. FIG. 14B depicts a bar graph of a human platelet adhesion assay to recombinant VWF proteins with and without the presence of a His-tag at a shear rate of 800 s⁻¹.

FIG. 15 represents models of the crystal structure of VWF-A1 domains solved using a recombinant protein. The main chain schematic of the mouse VWF-A1 domain, with β-strands (arrows) and helices (coils), is shown in FIG. 15A. The two cysteines involved in the disulfide bridge are shown as yellow spheres. FIG. 15B demonstrates that the C-alpha atoms of human (red) and mouse (blue) VWF-A1 domains closely overlap. FIG. 15C shows the model of the murine VWF-A 1 domain and the residues that purportedly interact with GPIb alpha, wherein amino acid residue 1326 of mouse (M) VWF-A1 was mutated to the corresponding amino acid at the identical location in its human counterpart (from Arg to His).

FIG. 16 shows graphs that depict platelet adhesion assays (FIG. 16A) and platelet translocation measurements (FIG. 16B). The ability of murine and human platelets to interact with a mutant protein substrate (human VWF-A1 domain wherein amino acid residue 1326 was mutated from His to Arg and mouse VWF-A1 domain wherein amino acid residue 1326 was mutated from Arg to His) was evaluated at a wall shear rate of 800 s⁻¹.

FIG. 17 represents data from an ELISA assay. Following several injections of mouse (M) VWF-A1, serum was collected from rats and screened by ELISA for anti-VWF-A1 antibodies. Spleens from animals with the highest antibody titers were harvested and splenocytes fused with Sp2/0 mouse myeloma cells (54). Supernatants of hybridomas were screened for reactivity to murine (M) VWF-A1 by ELISA. Pre-immune rat serum was used as control. Mabs to M VWF-A1 not only reacted with WT and mutant proteins (1324G>S) but also recognized native VWF purified from mouse plasma.

FIG. 18 shows representative graphs depicting the distribution of interaction times for more than 35 individual transient attachment events at various times. Analysis of the distribution of interactions times between human or murine VWF-A1 coated beads and their respective platelet substrates, as measured by high temporal resolution video microscopy, indicate that >95% of all transient tether bonds events fit a straight line, the regressed slope of which corresponded to a single k_(off), wherein the cellular off-rates of these quantal units of adhesion for the wild type human (H) and murine (M) proteins are found in FIGS. 18A and B and M VWF-A1 protein containing the type 2B mutation I1309V (1309I>V) corresponds to FIG. 18C.

FIG. 19 shows graphs that represent an assessment of transient tether events (FIG. 19A) and analysis of the distribution of interactions times (FIG. 19B) between human VWF-A1 coated microspheres and human immobilized platelets. The type 2B mutation Ile1309 Val (1309I>V) was incorporated into recombinant human (H) VWF-A1 containing either the type 2M mutation Gly1324Ser (1324G>S) or the function reducing mutation His1326Arg (1326H>R).

FIG. 20 is a scheme for generating transgenic mice with mutant VWF-A1 domains. FIG. 20A is a diagram of a knock-in construct for proposed mutations in the VWF-A1 domain of mice. FIG. 20B represents Southern blot hybridization with probe “a” or “b”, respectively, to determine if the construct was appropriately targeted.

FIG. 21 represents Southern blot analysis wherein heterozygous and homozygous mice for the amino acid substitution at residue 1326 (R1326H; 1326R>H) display the Arg1326His mutation (lanes 2 and 3 respectively) while wild-type animals did not (lane 1).

FIG. 22 represents sequence analysis of purified PCR products of WT, heterozygous, or homozygous VWF-A1 domains wherein the red-boxed area denotes the conversion of Arg to His (CGT in FIG. 22A wherein the codon corresponds to Arg and CAT in FIG. 22C wherein the codon corresponds to the amino acid His).

FIG. 23 is a graph of an ELISA assay which demonstrated that conversion of Arg to His in the mouse A1 domain did not alter plasma protein levels of VWF in mutant mice nor its ability to form multimers. The ELISA assay detected mouse VWF in plasma obtained from WT and homozygous (KI) animals, but not from plasma obtained from animals deficient in VWF (VWF KO).

FIG. 24 is a gel image of multimer gel analysis of plasma VWF that revealed an identical banding pattern between mouse and human VWF. Incorporation of His at position 1326 in the mouse A1 domain had no effect on multimerization of VWF in mutant mice.

FIG. 25 is a graphical representation of the bleeding times (s) observed in the mutant VWF-A1 mice that are either heterozygous or homozygous for the 1326R>H mutation. Results are compared to normal counterparts and VWF-deficient mice. Tail cut=1 cm.

FIG. 26 is a bar graph depicting thrombus formation induced by perfusion of whole blood from either wild type (WT) or homozygous mutant mice over surface-immobilized collagen in vitro wherein an 80% reduction in thrombus formation was observed compared to WT controls.

FIG. 27 are micrographs that demonstrate reduced thrombus formation occurring when whole blood from either the knock-in animals (homozygous for the R1326H mutation) or WT is perfused over collagen-coated cover slips at a shear rate of 1600 s⁻¹ indicating a 70% reduction in thrombi formed on collagen as compared to WT controls.

FIG. 28 demonstrates a platelet adhesion assay in flow. R1326H mutant mouse VWF promotes interactions with human platelets under physiologic flow conditions, wherein anticoagulated human blood was infused over surface-immobilized WT or mutant mouse plasma VWF at 1600 s⁻¹ as shown in the micrographs of FIG. 28A. FIG. 28B is a graph that depicts the amount of human platelets that bound to WT murine VWF or R1326H mutant murine VWF.

FIG. 29 are transmitted light micrographs demonstrating that homozygous mutant mice infused with human (FIG. 29A) but not mouse platelets (FIG. 29B) were able to generate an arterial thrombus that occludes the vessel lumen in response to laser-induced vascular injury as depicted by intravital microscopy.

FIG. 30 is a bar graph that depicts the average bleeding time for mice receiving blood-banked human platelets (˜3 minutes for a 1 cm tail cut) or given an intravenous infusion of a physiological buffered saline solution (10 minutes (end point)).

FIG. 31 is a schematic representing the isolation of the cremaster muscle and the catheter set-up used in intravital microscopy assays to assess thrombus formation.

FIG. 32 is a schematic of an intravital microscopy method.

FIG. 33 demonstrates images of mouse platelet interactions and a bar graph of such interactions in a wild type animal. FIG. 33A are representative intravital photomicrographs that depict the range of platelet interactions that occur at a site of vascular injury (60×). Platelets were observed to either transiently pause (*) or rapidly tether to and translocate (TP) on damaged arterial endothelium. A composite image demonstrates translocation of two platelets over a 3 s interval of time (panel 6). FIG. 33B depicts interacting platelets at the site of arterial injury that were classified as either undergoing translocation or firm adhesion (sticking) during an observation period of 1 min.

FIG. 34 represents photomicrographs that depict the vessel wall in a wild type mouse in the (A) absence of injury or (B) post-laser-induced injury as visualized under transillumination (40× objective). Thrombus is indicated by the arrows.

FIG. 35 is a graphical representation of the bleeding phenotype observed in the mutant VWF-A1 1326R>H heterozygous or homozygous mouse compared to its WT counterpart when tails were cut either 5 mm (FIG. 35A) or 15 mm (FIG. 35B) from the tip of the tail.

FIGS. 36A-B are graphs that depict ex vivo analysis of human platelet interactions with plasma VWF or recombinant VWF-A1 proteins. Accumulation of human platelets on surface-immobilized plasma human or mouse VWF (FIG. 36A) or recombinant human or mouse A1 domain proteins (FIG. 36B) after 4 min of perfusion with whole blood (shear rate of 1600 s⁻¹) is shown. Data are representative of three separate experiments performed in triplicate (mean±s.e.m.).

FIGS. 36C-D are graphs that depict ex vivo analysis of mouse platelet interactions with plasma VWF or recombinant VWF-A1 proteins. Accumulation of murine platelets on surface-immobilized human or mouse plasma VWF (FIG. 36C) or recombinant human or mouse A1 domain proteins (FIG. 36D) after 4 min of perfusion with whole blood (shear rate of 1600 s⁻¹) is shown. Data are representative of three separate experiments performed in triplicate (mean±s.e.m.).

FIGS. 37A-B are structural representations of human and murine VWF-A1 domains. FIG. 37A depicts the alignment of Cα atoms for human (blue) and murine (red) A1 domains. Key residues described in EXAMPLE 4 are shown as red spheres or as ball-and stick side-chains. FIG. 37B is a 90° rotation about a horizontal axis to reveal the packing of residue 1397 (Phe in human, Leu in mouse) that results in a 3 Å shift (blue arrow) of helix α4.

FIGS. 37C-D are structural representations of human and murine GPIbα-VWF-A1 complexes. FIG. 37C depicts the model of the murine-murine complex. FIG. 37D depicts the crystal structure of the human-human complex. Salt bridges are circled and key residue differences are boxed. Zooms reveal details of the electrostatic interactions at the β-switch contact region. The region of contact involving helix α3 of the A1 domain and one face of the LRR repeats of GPIbα is highly conserved between species, except for two residue changes that do not participate in bond formation: GPIbα E151K and VWF-A1 G1370S (human:mouse). Thus, minor differences in this region are unlikely to contribute to a reduction in binding between the murine and human proteins. This is also the case with the contact area located at the bottom of the A1 domain, which is invariant in both species and participates in salt-bridge formation (red circle).

FIG. 37E is a model of the human GPIbα-murine A1 complex, showing the loss (green arrow) and gain (blue circle) of salt-bridges. The upper zoom shows the interspecies interface at the β-switch region, revealing the electrostatic clash. The lower zoom shows the murine VWF-A1 point mutant 1326R>H, which removes the electrostatic clash and now closely resembles the human-human complex.

FIG. 37F is a model of the murine GPIbα-human VWF-A1 complex. Two salt-bridges are lost as compared to the murine complex; murine GPIbα D238 with residue 1326 due to the R>H change in human VWF-A1, and murine GPIbα K237 with residue 1330 owing to the E>G change in the human protein. Moreover, neither the chimeric nor murine complex forms a salt-bridge between residues 225 and 1395 on GPIbα and VWF-A1, respectively, as compared to its human counterpart (green circle). The upper zoom shows the interspecies interface at the β-switch region; there is no electrostatic clash but no salt-bridge can form with H1326. The lower zoom shows the human point mutant 1326H>R, which adds a salt-bridge and now closely resembles the murine-murine complex.

FIG. 37G is a graph that shows the accumulation of human platelets on surface-immobilized recombinant WT murine VWF-A1 domain proteins, those containing the selected mutations 1326R>H, 1330E>G and 1370S>G, or WT human VWF-A1 protein (shear rate of 1600 s⁻¹). Data are representative of three separate experiments performed in triplicate (mean±s.e.m.).

FIG. 38A is schematic for the generation of the VWF^(1326R>H) mouse that represents the targeting strategy for insertion of exon 28 containing adenine in lieu of guanine at position 3977 of the cDNA for murine VWF. R1, EcoRI; RV; EcoRV; B, BamHI; X, XhoI; pGK-TK, pGK-Neo, thymidine kinase/neomycin resistance cassette;

, loxP sites.

FIG. 38B is a blot of a Southern analysis of tailed DNA digested with EcoR1. Wild-type (WT) allele, 14 kb; mutant allele, 6 kb using Probe A. FIG. 38C represents the DNA sequencing of the tailed DNA demonstrating successful incorporation of adenine at position 3977 in heterozygous and homozygous animals (CGT>CAT). Sequence analysis of genomic DNA from these animals, 2 kb upstream and 6 kb downstream of exon 28, did not reveal any other alterations in nucleotide base pairs that would affect VWF production and/or function.

FIGS. 39A-B represent the analysis of VWF gene transcription and translation. FIG. 39A is a gel of RT-PCR of lung tissue from WT or mutant VWF mice to detect for A1, A2, and/or A3 domain message. β-actin analyzed to demonstrate use of equivalent amounts of mRNA. FIG. 39B is a graph demonstrating VWF antigen levels in plasma obtained from WT littermates (pooled) or six individual mice homozygous for 1326R>H mutation as detected by ELISA. Data are representative of two independent experiments performed in triplicate.

FIG. 39C is a gel showing the analysis of VWF multimers in plasma from WT or homozygous mutant animals. Normal human plasma as well as that obtained from a patient with type 2B VWD is shown for comparison.

FIG. 40 depicts representative photomicrographs showing murine platelet accumulation at sites of laser-induced arteriolar injury in WT or homozygous mutant animals 20 s and 2 min post-injury. White lines demarcate the extent of the thrombus.

FIG. 41A is a graphical representation of the tail bleeding times (s) for heterozygous and homozygous VWF^(1326R>H) and WT mice when tails were cut 1 cm from the tip of the tail. Each point represents one individual mouse and experiments were performed on five separate days.

FIG. 41B is an ex vivo analysis of human platelet interactions with surface-immobilized plasma VWF^(1326R>H) at a shear rate of 1,600 s⁻¹. A role for GPIb alpha on human platelets is demonstrated by the function-blocking antibody to this platelet receptor (mAb 6D1) to prevent adhesion in flow.

FIG. 41C are microscopy images of in vivo analysis of human platelet interactions with murine plasma VWF^(1326R>H) using infused fluorescently labeled human platelets into the vasculature of the cremaster muscle of mice. Human platelet accumulation was examined at sites of laser-induced arteriolar injury in WT (n=10) or homozygous mutant animals (n=12) using 2 channel confocal microscopy with excitation wavelengths of 488 nm (BCECF) and 561 nm (rhodamine 6G). Representative composite images of fluorescent images depicting human thrombus formation in homozygous mutant (upper panels) or WT (lower panels) mice (V=venule; A=arteriole). Rhodamine and BCECF are depicted in red and green, respectively, and merged is presented in yellow.

FIG. 41D is a bar graph depicting the composition of thrombi (% of total area) in WT or homozygous mutant animals.

FIG. 41E is a bar graph measuring thrombus size during an in vivo study of human platelet interactions with plasma VWF^(1326R>H) to determine the effect of GPIbα or αIIbβ3 blockade on human platelet adhesion in vivo. The requirement for GPIb alpha-mediated adhesion is shown by the ability of a function-blocking antibody (mAB 6D1 or mAb 7E3) to GPIb alpha to prevent human platelet thrombus formation in vivo. Fluorescently labeled human platelet accumulation was examined at sites of laser-induced arteriolar injury in WT (n=6) or homozygous mutant animals (n=8). Data represent the mean±s.e.m.

FIG. 41F is a graphical representation of tail bleeding times (s) for homozygous VWF^(1326R>H) that received an infusion of either normal saline or human platelets prior to severing 10 mm of distal tail, wherein the ability of human platelets to restore hemostasis in homozygous VWF^(1326R>H) and the effect of PLAVIX or ReoPro® on this process was examined. Each point represents one individual mouse and experiments were performed on 3 separate days.

FIG. 42 is a schematic depicting a perfluorocarbon nanoparticle capable of incorporating imaging agents (Gd⁺³, ^(99m)Tc) and chemotherapeutics into the outer layers. Antibodies complexed to the surface of the particle can target the agent to specific sites within the body.

FIG. 43 is a photographic image depicting the accumulation of fluorescent PNP, coupled to an antibody that recognizes human alphaIIb beta 3 on the surface of human platelets, at a site of vascular injury in homozygous 1326R>H mutant mice infused with human platelets.

FIG. 44 is a graphical representation of the structure of the VWF-A1-GPIb alpha-botrocetin ternary complex. FIG. 44A is a ribbon representation; GPIb alpha, green; botrocetin, red; A1, cyan. FIG. 44B demonstrates the location of the previously unknown interface between GPIb alpha and botrocetin.

FIG. 45A is a schematic representation showing that Recombinant GPIb alpha is surface-immobilized in a 96 well format. After blocking potential non-specific binding sites, recombinant VWF-A1 containing a His tag is added to the wells and allowed to interact with GPIb alpha for a specified period of time. The unbound material is removed by washing the wells and the complex formed between the 2 proteins detected by the addition of a HRP-conjugated antibody that binds to the His tag on A1. The amount of bound A1 can then be quantified by either fluorescence (addition of LumiGlow) or by color change.

FIG. 45B is an image representing that the specificity of the interaction can be determined by the addition of the GPIb function blocking antibody 6D1 prior to the addition of recombinant VWF-A1. DMSO (0.5%) was added to illustrate that this reagent does not interfere with the assay.

FIG. 46 are graphs depicting the effect of Plavix (FIG. 46A) or ReoPro (FIG. 46B) on human platelet-induced hemostasis in homozygous VWF^(1326R>H) mice.

FIG. 47 is a graph showing the efficacy of anti-platelet drugs administered to patients by studying the ability of platelets harvested from patients on therapies in the VWF^(1326R>H) mouse.

SUMMARY OF THE INVENTION

The invention provides for a mouse model for pre-clinical screening and testing of candidate compounds, wherein the model comprises a mouse expressing plasma von Willebrand Factor protein that contains a mutation or combination of mutations in its A1 domain that change the mouse protein's binding specificity from being specific for mouse platelets to being specific for human platelets. Thus, the mutant A1 domain contained within mouse plasma VWF particularly supports the binding of human platelets in vivo and ex-vivo. In one embodiment, the mouse model is infused with human platelets. In another embodiment, human platelets are labeled ex-vivo or in vivo so as to be detected while in the animal.

The invention provides for where the mutation in the A1 domain of mouse VWF comprises 1263P>S, 1269N>D, 1274K>R, 1287M>R, 1302G>D, 1308H>R, 1313R>W, 1314I>V, 1326R>H, 1329L>1,1330E>G, 1333A>D, 1344T>A, 1347I>V, 1350T>A, 1370G>S, 1379H>R, 1381T>A, 1385T>M 1391P>Q, 1394A>S, 1397L>F, 1421S>N, 1439L>V, 1442G>S, 1449R>Q, 1466A>P, 1469Q>L, 1472Q>H, 1473V>M, 1475H>Q, 1479S>G, and any combination thereof, wherein the mutation corresponds to an amino acid position of human von Willebrand Factor A1 protein shown in SEQ ID NO: 6. In addition, the mouse VWF A1 domain can be fully or partially replaced with the human VWF A1 domain. In one embodiment, the mutation in mouse VWF-A1 is 1326R>H. In another embodiment, the mouse model comprises a device within a vessel, such as a stent or a graft, or mechanical, chemical, or heat-induced disruption of vascular endothelium in vivo. This model system is useful for testing compounds in an in vivo environment. The compounds can be tested for an effect on the interaction between human platelets and human-like (the mutant VWF-A1, 1326R>H, for example), or the actual human VWF-A1 domain. For example, the animal model can be used for pre-clinical testing of drugs in order to determine whether 1) there is a desired effect on hemostasis and/or thrombus formation or anti-thrombotic effect by the test drug or 2) there is an undesired effect on hemostasis and/or thrombus formation or anti-thrombotic effect by a test drug not specifically designed to alter hemostasis and/or thrombus formation. In the latter case, many drugs are only identified as having an effect on clotting or bleeding once they are in human clinical trials, this animal model will fill an unmet need, which is to test such effects prior to clinical trials. The invention also permits testing of compounds targeted to the VWF-A1 domain that can correct the bleeding phenotype associated with a loss-of-function mutations (1326R>H, for example) by altering the kinetics of the interaction between GPIbα and VWF-A1 (for example, enhancing the on-rate and/or prolonging the bond lifetime as shown for the snake venom protein botrocetin.

The invention also provides for an isolated mutant human von Willebrand Factor A1 protein comprising one or more mutations selected from the group consisting of: 1263S>P, 1269D>N, 1274R>K, 1287R>M, 1302D>G, 1308R>H, 1313W>R, 1314V>I, 1326H>R, 1329I>L, 1330G>E, 1333D>A, 1344A>T, 1347V>I, 1350A>T, 1370S>G, 1379R>H, 1381A>T, 1385M>T, 1391Q>P, 1394S>A, 1397F>L, 1421N>S, 1439V>L, 1442S>G, 1449Q>R, 1466P>A, 1469L>Q, 1472H>Q, 1473M>V, 1475Q>H, 1479G>S, wherein each amino acid position corresponds to a position in SEQ ID NO: 6. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1263S>P mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1269D>N mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1274R>K mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1287R>M mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1302D>G mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1308R>H mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1313W>R mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1314V>I mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1326H>R mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1329I>L mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1330G>E mutation in an amino acid sequence of SEQ ID NO:1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1333D>A mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1344A>T mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1347V>I mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1350A>T mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1370S>G mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1379R>H mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1381 A>T mutation in an amino acid sequence of SEQ ID NO:1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1385M>T mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1391Q>P mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1394S>A mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1397F>L mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1421N>S mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1439V>L mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1442S>G mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1449Q>R mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1466P>A mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1469L>Q mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1472H>Q mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1473M>V mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1475Q>H mutation in an amino acid sequence of SEQ ID NO: 1. The invention also provides for an isolated mutant human von Willebrand Factor A1 protein consisting of a 1479G>S mutation in an amino acid sequence of SEQ ID NO: 1.

The invention also provides for an isolated mutant human von Willebrand Factor A1 protein having SEQ ID NO: 6, wherein the protein comprises a mutation selected from the group consisting of: 1263S>P, 1269D>N, 1274R>K, 1287R>M, 1302D>G, 1308R>H, 1313W>R, 1314V>I, 1326H>R, 1329I>L, 1330G>E, 1333D>A, 1344A>T, 1347V>I, 1350A>T, 1370S>G, 1379R>H, 1381A>T, 1385M>T, 1391Q>P, 1394S>A, 1397F>L, 1421N>S, 1439V>L, 1442S>G, 1449Q>R, 1466P>A, 1469L>Q, 1472H>Q, 1473M>V, 1475Q>H, or a 1479G>S.

The invention provides for a transgenic non-human animal expressing von Willebrand Factor A1 protein containing mutation(s) at one of more amino acid position selected from the group consisting of: 1263, 1269, 1274, 1287, 1302, 1308, 1313, 1314, 1326, 1329, 1330, 1333, 1344, 1347, 1350, 1370, 1379, 1381, 1385, 1391, 1394, 1397, 1421, 1439, 1442, 1449, 1466, 1469, 1472, 1473, 1475, and 1479, wherein the position corresponds to an amino acid position of human von Willebrand Factor A1 protein shown in SEQ ID NO: 6. In one embodiment, the animal is a murine, a porcine, a canine, a feline, a rabbit, or a primate. In another embodiment, the animal is a mouse, a rat, a dog, a sheep, a goat, a horse, a cow, a cat, a monkey, a primate, a pig, a llama, an alpaca, a chicken, etc. In other embodiments, the protein comprises a single mutation. In further embodiments, the protein comprises two or more mutations. In yet another embodiment, the protein comprises at least one mutation selected from the group consisting of: 1263>S, 1269>D, 1274>R, 1287>R, 1302>D, 1308>R, 1313R>W, 1314>V, 1326>H, 1329>I, 1330>G, 1333>D, 1344>A, 1347>V, 1350>A, 1370>S, 1379>R, 1381>A, 1385>M 1391>Q, 1394>S, 1397>F, 1421>N, 1439>V, 1442>S, 1449>Q, 1466>P, 1469>L, 1472>H, 1473>M, 1475>Q, 1479>G, and any combination thereof. In particular embodiments, the protein comprises a 1326R>H mutation. In other embodiments, the protein comprises a 1314I>V mutation. In yet other embodiments, the protein comprises a 1326R>H mutation, a 1314I>V mutation, or a combination of the two mutations listed previously. In some embodiments of the invention, the animal is a mouse. In further embodiments, the protein comprises SEQ ID NO: 5. In other embodiments, the VWF protein is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the A1 domain of human VWF protein as shown in SEQ ID NO: 1. In particular embodiments, the von Willebrand Factor A1 protein of the transgenic animal comprises the human A1 domain shown in SEQ ID NO: 1. In some embodiments, the von Willebrand Factor A1 protein is partially or completely replaced with a human von Willebrand Factor A1 protein comprising SEQ ID NO: 1. In other embodiments, the animal is a model for pre-clinical testing of compounds that expresses a mutant von Willebrand Factor (VWF) A1 protein containing one or more mutations, wherein the binding specificity of the mutant VWF-A1 protein changes from being specific for the animal platelets to being specific for human platelets. In further embodiments the mutant VWF-A1 protein in the animal binds to human platelets.

The invention is provides a method for identifying a compound that modulates binding of VWF-A1 protein to GPIb-alpha protein. The method comprises: providing an electronic library of test compounds; providing atomic coordinates listed in Table 8 for at least 10 amino acid residues for the A1 domain of the VWF protein, wherein the coordinates have a root mean square deviation therefrom, with respect to at least 50% of Cα atoms, of not greater than about 2.5 Å, in a computer readable format; converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the VWF-A1 domain; performing a data processing method, wherein electronic test compounds from the library are superimposed upon the three dimensional model of the VWF-A1 domain; and determining which test compound fits into the binding pocket of the three dimensional model of the VWF-A1 protein, thereby identifying which compound would modulate the binding of VWF-A1 protein to GPIb-alpha protein. Alternatively, the method can comprise: providing an electronic library of test compounds; providing atomic coordinates listed in Table 8 in a computer readable format for at least 10, 15, 20, 25, 30, 35, or 40 amino acid residues for the A1 domain of the VWF protein, wherein the residues comprise two or more of the following residues: Pro1391, Arg1392, Arg1395, Val1398, Arg1399, Gln1402, Lys1406, Lys1423, Gln1424, Leu1427, Lys1430, or Glu1431; converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the VWF-A1 domain; performing a data processing method, wherein electronic test compounds from the library are superimposed upon the three dimensional model of the VWF-A1 domain; and determining which test compound fits into the binding pocket of the three dimensional model of the VWF-A1 protein, thereby identifying which compound would modulate the binding of VWF-A1 protein to GPIb-alpha protein.

In one embodiment, determining comprises detecting an IC₅₀ of less than about 7.5 μg/ml for a test compound. In another embodiment, the method can further comprise: obtaining or synthesizing a compound; contacting VWF-A1 protein with the compound under a condition suitable for GPIb-alpha-VWF-A1 binding; and determining whether the compound modulates GPIb-alpha-VWF-A1 protein binding using a diagnostic assay. In a further embodiment, contacting comprises perfusing platelets into a flow chamber at a shear flow rate of at least 100 s⁻¹, wherein mutant murine VWF-A1 protein is immobilized on a bottom surface of the chamber, while in another embodiment contacting comprises perfusing platelets into the transgenic non-human animal described above. In some embodiments, contacting occurs sequentially. In other embodiments, the perfusing of platelets occurs prior to administration of the compound. In one embodiment, the platelets are human platelets, while in other embodiments, the platelets are not murine platelets. In further embodiments, the determining comprises detecting an increase or decrease in the dissociation rate between VWF-A1 protein and GPIb-alpha protein by at least two-fold. In other embodiments, the determining comprises detecting an increase or decrease of platelet adhesion to a surface expressing VWF-A1 protein, while in some embodiments the determining comprises detecting an increase or decrease in a stabilization of an interaction between VWF-A1 protein and GPIb-alpha protein. In particular embodiments, the determining comprises detecting thrombosis formation. In some embodiments, the determining comprises identifying an occurrence of an abnormal thrombotic event in the subject. In further embodiments of the invention, an abnormal thrombotic event comprises abnormal bleeding, abnormal clotting, death, or a combination of the events listed. In some embodiments, the determining comprises dynamic force microscopy, a coagulation factor assay, a platelet adhesion assay, thrombus imaging, a bleeding time assay, aggregometry, review of real-time video of blood flow, a Doppler ultrasound vessel occlusion assay, or a combination thereof. In particular embodiments of the invention, perfusing platelets is followed by perfusion of a labeled agent. In some embodiments, the labeled agent comprises one or more of a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, or a small molecule ligand. In other embodiments, the agent targets a platelet receptor, a VWF protein, or a portion thereof. In another embodiment, the animal is injected with nanoparticles, and/or peptides, and/or small molecules, which label the human platelets, at some time prior testing, wherein the nanoparticles, and/or peptides, and/or small molecules are capable of being imaged while in the animal. In another embodiment, the testing comprises tracking of human platelet deposition in the animal. In another embodiment, the compound or agent is an anti-thrombotic, such as an anti-platelet drug, e.g., PLAVIX, an ADP inhibitor, and/or a humanized antibody and/or small molecule that inhibits human alpha IIb and/or beta 3 integrin function, human alpha2 and/or beta 1 integrin function, human glycoprotein VI (GPVI) function, human thrombin receptors function, and/or intracellular signaling pathways (for example, phosphoinositide 3-kinases (PI3K)) vital to platelet function in hemostasis and thrombosis.

The invention provides for a method for testing a compound or agent, the method comprising: (a) providing a candidate agent or compound to be tested; (b) administering the agent or compound to an animal in an effective amount, wherein the animal expresses a mutant von Willebrand Factor A1 protein containing a mutation, combination of mutations that change the animal protein's binding specificity from being specific for animal platelets to being specific for human platelets, so that the mutant VWF-A1 protein in the animal binds to human platelets, and wherein the animal is perfused with human platelets; (c) testing the animal to determine whether the animal experiences any abnormal hemostatic and/or thrombotic events, thereby testing the compound or agent.

The invention provides for a nucleic acid encoding the mutant von Willebrand Factor A1 protein of the invention. The invention provides for a vector containing such a nucleic acid. The invention provides for an animal expressing such a nucleic acid to express the mutant VWF protein.

The invention also provides a method for treating von Willebrand Disease (VWD) in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound that promotes platelet adhesion in the subject, wherein the compound increases the dissociation rate between VWF-A1 protein and GPIb-alpha protein by at least two-fold, thereby administration of the compound increases blood coagulation in the subject. In one embodiment, coagulation is measured by a coagulation factor assay, an ex-vivo flow chamber assay, or a combination thereof.

The invention provides a method for rapidly detecting an internal vascular injury site in a subject. The method comprises: administering to a subject a targeted molecular imaging agent, wherein the molecule circulates for an effective period of time in order to bind to the injury site within the subject; tracking a deposition of the labeled targeted molecular imaging agent in the subject; and identifying the site of a thrombus formation in the subject by imaging the targeted molecular imaging agent, thereby the deposition of the targeted molecular imaging agent at the internal vascular injury site is indicative of internal bleeding within a subject. In one embodiment, the targeted molecular imaging agent is administered by subcutaneous, intra-muscular, intra-peritoneal, or intravenous injection; infusion; by oral, nasal, or topical delivery; or a combination of the routes listed. In another embodiment, the targeted molecular imaging agent comprises a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, a peptide, a small molecule ligand, or a combination of the agents listed. In a further embodiment, the nanoparticle comprises a perfluorocarbon, while in particular embodiments, the nanoparticle is coupled to an antibody, a small molecule, a peptide, or a receptor trap. In some embodiments, the targeted molecular imaging agent specifically binds to a platelet receptor, or a VWF protein, or a portion thereof. In other embodiments, the targeted molecular imaging agent has a T_(1/2) of at least 30 minutes. In further embodiments, imaging comprises a PET scan, MRI, IR scan, ultrasound, nuclear imaging, or a combination of the methods listed. In a particular embodiment, the subject is further administered a pro-thrombotic compound. In a further embodiment, the compound increases the dissociation rate between VWF-A1 protein and GPIb-alpha protein by at least two-fold.

Considerable emphasis has been placed on elucidating the role of structural changes in the A1 domain of VWF in order to gain insight into mechanism(s) that may regulate its binding to platelet GPIb alpha. This invention concerns the contribution of the biophysical properties of this interaction in governing platelet adhesion under hydrodynamic conditions. It has been demonstrated that flow-dependent adhesion and rapid and force-driven kinetic properties define the GPIb alpha-VWF-A1 bond. The invention provides classification of subtypes of von Willebrand disease (vWD), such as platelet-type, type 2B or type 2M in terms of similarities in the alterations in the biophysical properties of bonds to better understand the clinical phenotypes associated with these bleeding disorders. The invention is directed to understanding the biomechanical and molecular mechanisms by which the VWF-A1 domain mediates adhesive interactions with GPIb alpha both in vitro and in vivo. This invention provides for mice with mutant A1 domains to demonstrate the importance of the intrinsic kinetic and mechanical properties of this receptor-ligand pair in preventing inappropriate platelet aggregation in circulating blood, to facilitate the study of human platelet biology, as well as to generate a humanized animal model of hemostasis and thrombosis. The animal model is also useful for the generation and testing of novel anti-thrombotic therapies designed to inhibit platelet-VWF interactions as well as those designed to correct the bleeding phenotype associated with a reduction in adhesion between this receptor-ligand pair.

The invention is directed to understanding the effect that alterations in platelet size and shape have on the force-driven kinetics of the GPIb alpha-VWF-A1 tether bond. Platelets can bind to and translocate on surface-immobilized VWF under shear forces that preclude selectin-dependent adhesion of leukocytes to the vessel wall, due to their small discoid shape and not as a consequence of the unique kinetic properties of the GPIb alpha-VWF-A1 tether bond. The contribution of particle geometry in supporting the interactions between this receptor-ligand pair, we is performed by evaluating the interaction between vWF-A1 coated microspheres, ranging from 4 to 12 μm in diameter (platelet to leukocyte size), with surface-immobilized platelets under physiologic flow conditions. The utility of using polystyrene microspheres with a uniform shape and size to permit determination of the relationship between wall shear stress and the force acting on the GPIb alpha-vWF-A1 tether bond has been demonstrated. Moreover, it has been shown that the “sidedness” of the receptor or ligand does not alter the kinetic properties of this bond. β-tubulin deficient mice have a defect in the cytoskeleton of platelets that changes the shape of these cells from discoid to spherical. Preliminary data demonstrates that the “spherical” platelets have a >60% reduction in attachment at high shear rates as compare to wild-type (WT) platelets. No differences in the kinetics of the GPIb alpha-vWF-A1 tether bond for WT and mutant platelets have been found using our microsphere assay system. Thus, platelet shape and not alterations in the biophysical properties of the GPIb alpha-vWF-A1 tether bond are responsible for the defect in adhesion. Data generated from these experiments will be use to develop a computational algorithm designed to stimulate the adhesion of platelets to surface-immobilized vWF under various hydrodynamic conditions. Thus, the invention provides an in vivo method to test for defects in hemostasis and thrombus formation that result from abnormalities in platelet shape and size. It also provides for a method to test the ability of synthetic platelet substitutes, which may be of varying shapes and sizes, to support hemostasis.

The invention provides a method for determining whether platelet function or morphology in a subject is abnormal. The method comprises: affixing a molecule comprising a murine VWF-A1 domain to a surface of a flow chamber, wherein the domain comprises at least one mutation at a position selected from the group consisting of 1263>S, 1269>D, 1274>R, 1287>R, 1302>D, 1308>R, 1313R>W, 1314>V, 1326>H, 1329>I, 1330>G, 1333>D, 1344>A, 1347>V, 1350>A, 1370>S, 1379>R, 1381>A, 1385>M 1391>Q, 1394>S, 1397>F, 1421>N, 1439>V, 1442>S, 1449>Q, 1466>P, 1469>L, 1472>H, 1473>M, 1475>Q, 1479>G, and any combination thereof, where the position corresponds to an amino acid position of human von Willebrand Factor A1 protein shown in SEQ ID NO: 6; perfusing through the flow chamber a volume of blood or plasma from a subject at a shear flow rate of at least about 100 s⁻¹; perfusing a targeted molecular imaging agent into the flow chamber; and comparing the flow rate of the blood or plasma from the subject as compared to a normal flow rate, so as to determine whether the subject's platelet function or morphology is abnormal.

In one embodiment, affixing comprises (i) affixing an antibody which specifically binds VWF-A1 domain, and (ii) perfusing murine mutant VWF-A1 protein in the flow chamber at a shear flow rate of at least 100 s⁻¹. In another embodiment, the targeted molecular imaging agent comprises a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, a peptide, a small molecule ligand, or a combination of the agents listed. In a further embodiment, the targeted molecular imaging agent binds to a platelet receptor, a platelet ligand, or any region of a VWF protein or a portion thereof. In a particular embodiment, the targeted molecular imaging agent comprises horseradish peroxidase (HRP) coupled to an antibody directed at VWF-A1. In other embodiments, the comparing comprises a platelet adhesion assay, fluorescence imaging, a chromogenic indicator assay, a microscopy morphology analysis, or any combination of the listed modes. In some embodiments, platelets bound to VWF-A1 are less than about 500 cells/mm². In particular embodiments, the platelets are substantially spherical. In further embodiments, the subject is a human, a canine, a feline, a murine, a porcine, an equine, or a bovine. In other embodiments, the VWF molecule is an antibody, a peptide, or a Fab fragment directed to a VWF polypeptide or a portion thereof.

The invention also provides for a method for producing von Willebrand Factor A1 protein that specifically binds human platelets, the method comprising: (a) providing an animal expressing a plasma VWF containing a mutant von Willebrand Factor A1 protein, wherein the mutation causes the animal's von Willebrand Factor A1 protein to bind preferentially to for human platelets; and (b) harvesting the mutant animal von Willebrand Factor A1 so as to produce von Willebrand Factor A1 protein that specifically binds human platelets. In one embodiment, the mutant animal von Willebrand Factor A1 protein comprises at least one mutation comprising 1263P>S, 1269N>D, 1274K>R, 1287M>R, 1302G>D, 1308H>R, 1313R>W, 1314I>V, 1326R>H, 1329L>I, 1330E>G, 1333A>D, 1344T>A, 1347I>V, 1350T>A, 1370G>S, 1379H>R, 1381T>A, 1385T>M 1391P>Q, 1394A>S, 1397L>F, 1421S>N, 1439L>V, 1442G>S, 1449R>Q, 1466A>P, 1469Q>L, 1472Q>H, 1473V>M, 1475H>Q, 1479S>G, or any combination thereof.

The invention provides a method for testing efficacy and toxicity of a gene therapy vector, wherein the method comprises: (a) introducing a gene therapy vector into the animal of claim 4, allowing sufficient time for expression of the vector; (b) perfusing platelets from a subject into the animal under a condition suitable for GPIb-alpha-VWF-A1 protein binding; and (c) determining whether or not a thrombotic event occurs in the animal. In one embodiment, the vector comprises a nucleic acid encoding a platelet receptor polypeptide, a platelet ligand polypeptide, or a VWF polypeptide, or a portion thereof. In another embodiment, the subject is a human, a dog, a cat, a horse, a pig, or a primate. In a particular embodiment, the platelets are not murine platelets. In a further embodiment, the thrombotic event comprises blood clotting, abnormal bleeding, abnormal clotting, death, or a combination thereof. In some embodiments, the determining comprises dynamic force microscopy, a coagulation factor assay, a platelet adhesion assay, thrombus imaging, a bleeding time assay, aggregometry, review of real-time video of blood flow, a Doppler ultrasound vessel occlusion assay, or a combination thereof. In other embodiments, perfusing platelets is followed by perfusion of a labeled agent. In further embodiments, the labeled agent comprises one or more of a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, or a small molecule ligand. In particular embodiments, the agent targets a platelet receptor, a VWF protein, or a portion thereof.

The invention also provides a method for calibrating an aggregometry device or a device for measuring clot formation or retraction, wherein the method comprises: (a) providing hematologic data obtained from a subject, wherein blood or platelets from the subject is assessed by the device; (b) determining whether or not a thrombotic event occurs in the animal described above, wherein the animal is perfused with a sample of blood or platelets from the subject; and (c) correlating data obtained from (b) with the data obtained in (a) so as to calibrate the device, wherein a certain data obtained from the device is indicative of the corresponding thrombotic outcome determined in the animal described above. In one embodiment, the thrombotic event comprises blood clotting, abnormal bleeding, abnormal clotting, death, or a combination thereof.

The invention provides for in vivo determination of intrinsic and mechanical properties of the GPIb alpha-vWF-A1 tether bond and to determine if they are indeed critical for regulating the adhesion between platelets and vWF and how they may be manipulated to impair or enhance hemostasis and/or thrombosis. The invention provides determination of whether animals that posses gain-of-function mutations in VWF-A1, for example, those associated with type 2B vWD, have a defect in platelet deposition at sites of vascular injury and/or a loss of high molecular weight multimers of vWF. The invention provides determination of whether animals that possess loss-of-function mutations in VWF-A1, for example, those associated type 2M vWD, have a defect in platelet deposition at sites of vascular injury. Based on the results obtained herein, mice are genetically engineered with 1) mutant A1 domains that increase or decrease the on- and/or off-rate of this receptor-ligand pair by varying degrees, 2) A1 domains containing both types of mutations to confirm whether specific regions within this domain are essential for the stabilization of GPIb alpha binding, and 3) mutations within the A1 domain that favor binding to human but not murine GPIb alpha to enable the study of human platelet behavior in an animal model of hemostasis or thrombosis.

The invention provides methods for determining the impact of altering the intrinsic bond kinetics and/or its mechanical properties of the GPIb alpha-VWF-A1 interaction on hemostasis and thrombosis, which comprises: measuring platelet counts, plasma levels of vWF, and bleeding times; performing multimer gel analysis of mutant vWF; measuring the affinity of mutant vWF for platelets using a fluid phase binding assay; evaluating in vitro platelet tethering, rolling, and thrombus formation on surface-immobilized murine plasma vWF containing mutant A1 domains under physiological flow conditions; determining the ability of thrombi to form at sites of vascular injury in vivo in mutant VWF mice using epifluorescent intravital microscopy in; measuring platelet tethering frequency and rolling velocities in vivo.

In certain embodiments, the subject is a human. In other embodiments, the subject is a non-human animal such as a canine, equine, feline, porcine, murine, bovine, foul, sheep, or any other animal in need of treatment. In certain embodiments, the pharmaceutical composition further comprises another active agent. The additional active agent can be, but is not limited to, an analgesic, an antioxidant, diuretic, or a combination thereof. In certain embodiments, the composition is in a capsule form, a granule form, a powder form, a solution form, a suspension form, a tablet form, or any other form suitable for use by the method of the present invention. In certain embodiments, the composition is administered via oral, sublingual, buccal, parenteral, intravenous, transdermal, inhalation, intranasal, vaginal, intramuscular, rectal administration or any other route of administration that is suitable for delivery of the compound.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods for identifying and evaluating potential anti-thrombotic reagents and compounds. The invention provides methods for testing for undesirable thrombotic or bleeding side effects of reagents in the setting of preclinical testing. The invention provides an in vivo model to test the efficacy of potential anti-thrombotic drugs directed against receptors, ligands, and/or intracellular signaling pathways on or in human platelets prior to FDA approval. To date, in vitro models of thrombosis do not accurately recapitulate the hemodynamic conditions, cell-cell interactions, or cell-protein interactions that occur at sites of vascular injury in a living animal. Thus, the system provided by this invention provides a method to test drugs directed at inhibiting or altering human platelet function other than directly testing them in humans. The invention provides a great advantage of being able to test directly compounds that target human platelets in an in vivo system. The invention provides a method to test contrast agents for imaging of human platelets at sites of thrombosis. For instance, one could test the ability of nanoparticle contrast agents targeted to human platelets to identify areas of thrombosis or occult bleeding. The invention provides a method to test compounds that correct the bleeding phenotype associated with a reduction in interactions between GPIb alpha and VWF-A1. The invention provides a method to test gene therapies directed at correcting genetic mutations associated with von Willebrand disease. The invention provides a method to correlate results obtained with an in vitro assay designed to measure the effects of antithrombotics or markers of platelet activation in patients.

Terms

A “pharmaceutical composition” refers to a mixture of one or more of the compounds, or pharmaceutically acceptable salts, hydrates, polymorphs, or pro-drugs thereof, with other chemical components, such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

As used herein von Willebrand factor is abbreviated “VWF” and, alternatively, “vWF”.

A “pro-drug” refers to an agent which is converted into the parent drug in vivo. Pro-drugs are often useful because, in some situations, they are easier to administer than the parent drug. They are bioavailable, for instance, by oral administration whereas the parent drug is not. The pro-drug also has improved solubility in pharmaceutical compositions over the parent drug. For example, the compound carries protective groups which are split off by hydrolysis in body fluids, e.g., in the bloodstream, thus releasing active compound or is oxidized or reduced in body fluids to release the compound.

A compound of the present invention also can be formulated as a pharmaceutically acceptable salt, e.g., acid addition salt, and complexes thereof. The preparation of such salts can facilitate the pharmacological use by altering the physical characteristics of the agent without preventing its physiological effect. Examples of useful alterations in physical properties include, but are not limited to, lowering the melting point to facilitate transmucosal administration and increasing the solubility to facilitate administering higher concentrations of the drug.

The term “pharmaceutically acceptable salt” means a salt, which is suitable for or compatible with the treatment of a patient or a subject such as a human patient or an animal.

The term “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any base compounds of the invention or any of their intermediates. Illustrative inorganic acids which form suitable acid addition salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable acid addition salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed and such salts exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of compounds of the invention are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts, e.g., oxalates, are used, for example, in the isolation of compounds of the invention for laboratory use or for subsequent conversion to a pharmaceutically acceptable acid addition salt.

As used herein and as well understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

Arterial thrombosis is a pathological consequence of disease states such as atherosclerosis and remains a major cause of morbidity and mortality in the Western world with healthcare cost ranging in the billions of dollars in the USA alone (Circulation 2006; 113:e85). Central to this process is the inappropriate deposition and activation of platelets in diseased vessels that can ultimately occlude the lumen, thus impeding blood flow to vital organ such as the heart and brain. Another key player is VWF, a large plasma glycoprotein of complex multimeric structure, which under normal physiological conditions prevents excessive bleeding by promoting platelet deposition at sites of vascular injury, thus “sealing off” leaky blood vessels. In order for this event to occur, VWF must form a “bridge” between receptors expressed on circulating platelets and exposed components of the injured vessel wall. This is the function of the A1 and A3 domains of this plasma protein, respectively. Each is folded into a disulfide-bonded loop structure that is critical for optimal biological activity (FIG. 1A).

It is the A1 domain that contains residues that compose the binding site for its receptor on platelets known as GPIb alpha, an adhesive event essential for the ability of these cells to rapidly attach to the injured vessel wall. The critical nature of this interaction is exemplified by the bleeding disorder, termed type 2M von Willebrand Disease (VWD), which results from the incorporation of loss-of-function point mutations within this domain that reduce the interaction between VWF-A1 and GPIb alpha (Sadler J E et al.(2006) J. Thromb. Haemost. 4: 2103-14). The A3 domain, on the other hand, is believed to be important in anchoring plasma VWF to sites where extracellular matrix components (i.e. collagen) are exposed as a result of disruption of the overlying vasculature endothelium (Wu D. et al. Blood 2002). Once in contact with exposed elements of the damaged vessel wall, platelets become “activated” through various signaling pathways (i.e. GPVI) enabling other adhesion molecules, such as α2β1 (collagen receptor) and αIIbβ3 (fibrinogen and VWF receptor) integrins, to firmly anchor these cells at the site of injury and to each other (FIG. 1B). In addition, ADP released from adherent platelets serves to amplify the activation of integrin receptors as well as other platelets leading to thrombus growth. Considerable emphasis has been placed on understanding the mechanism(s) that govern the interaction between GPIb alpha and the A1 domain of VWF and how it can be perturbed by point mutations associated with von Willebrand Disease, information relevant to the development of anti-thrombotic therapies.

von Willebrand Factor (VWF), the A1 Domain, and Related Diseases

During the past two decades, there has been considerable progress in understanding how VWF mediates platelet adhesion. Both the VWF cDNA and gene have been cloned and the primary structure of the VWF subunit (FIG. 1A) has been determined (13-16). It has been reported that ˜59% of the mature VWF consists of repeated segments which are 29% to 43% homologous (17). These regions consist of domains that are triplicated (domains “A” and “B”), duplicated (domain “C”) or quadruplicated (domain “D”). The triplicated A repeats encode for the central region of each VWF subunit. The A1 and A3 domains contain the sequences that mediate VWF's interaction with receptors on platelets or components of subendothelial extracellular matrix, respectively. Each is folded into a disulfide-bonded loop structure that is critical for optimal biological activity. The sequences of the amino terminal halves of each loop and the location of the cysteines forming the loop structure of each domain are highly conserved. It is the VWF-A1 domain (1260-1480) that contains sequences that provide binding sites for the platelet glycoprotein receptor Ib alpha, an interaction critical for the ability of these cells to rapidly attach and translocate at sites vascular injury (18,19). The role of the A3 domain, however, is believed to be in anchoring plasma VWF at sites where extracellular matrix components (i.e. collagen) are exposed as a result of disruption of the endothelium (20-25).

With regard to mediating adhesive interactions with platelets, it has become increasingly evident that the VWF-A1 domain plays a crucial role in this process based on molecular genetic studies of individuals with type 2M or 2B VWD (26-31). In the majority of cases, patients with these designated genotypes have single point mutations contained within the disulfide loop (between Cys 1272 and Cys 1458) of this domain. With regard to type 2M VWD, afflicted individuals have significant impairments in hemostasis that appears to result from a lack of or reduced adhesive interactions between GPIb alpha and VWF at sites of vascular injury, and not from an alteration in VWF multimer structure. Structural and functional evidence has been provided in that type 2M mutations, such as 1324G>S, are localized within a region of the A1 domain (FIG. 5B), which is critical for supporting GPIb alpha-mediated platelet adhesion at physiological flow rates. Confirmation that this residue, as well as others predicted by our analysis of the crystal structure of the A1 domain, does contribute to GPIb alpha binding is suggested by studies evaluating the structure of the complex formed between this receptor-ligand pair (32, 33). Thus, it is possible to make accurate predictions about protein function from the three-dimensional protein structure and to confirm these postulates by site-specific mutagenesis and analysis under physiologically relevant flow conditions. The localization of some of the residues within the A1 domain that when mutated disrupt GPIb alpha binding is shown below (FIG. 2). The invention provides methods for evaluating the effect that loss-of-function mutations have on hemostasis and thrombus formation.

In contrast type 2M VWD, mutations associated with type 2B VWD are known to enhance the interaction between VWF-A1 and GPIb alpha, that is, they mitigate the requirement for exogenous modulators such as ristocetin or botrocetin to induce platelet agglutination (30). Moreover, these altered residues are localized in a region remote from the major GPIb alpha binding site that has been identified by mutagenesis (26; FIG. 3A—yellow). Clinically, this disease state is characterized by a loss of circulating high molecular weight VWF multimers (HMWM, FIG. 3B) together with a mild to moderate thrombocytopenia, which ultimately results in bleeding but not thrombosis (30, 31). It is the clearance of the HMWM of VWF from plasma that is believed to be responsible for the increased bleeding tendencies in patients with this disorder as they contribute to the majority of the hemostatic function associated with this plasma glycoprotein (34). The invention provides methods for evaluating the effect that gain-of-function mutations have on hemostasis, thrombus formation, and plasma levels of VWF.

Surface-immobilization of VWF and subsequent exposure to physiologically relevant shear forces appears to be a prerequisite for its ability to support interactions with platelets as this multimeric protein does not bind appreciably to these cells in the circulation. These hydrodynamic conditions are believed to promote structural changes within the A1 domain that in turn increases its affinity for GPIb alpha (35-37). Evidence suggested to support the existence of such an alteration in structure includes the ability of non-physiologic modulators such as the antibiotic ristocetin or the snake venom protein botrocetin to promote platelet agglutination in solution-based assays (38, 39). Moreover, this “on” and “off” conformation is exemplified by type 2B VWD. For instance, it was initially hypothesized that incorporation of type 2B mutations into the A1 domain shifted the equilibrium between two distinct tertiary conformations, analogous to those seen in crystal structures of the integrin I domain in ligand-free and collagen-bound states (40). The location of the type 2B mutants at sites distinct from the GPIb alpha binding site suggest that they disrupt a region responsible for regulation of binding affinity, thus affecting ligand binding allosterically. The crystal structure of a type 2B mutant A1 domain, 1309I>V, was determined and compared to its wild-type counterpart (41). A change was discovered in the structure of a loop, thought to be involved in GPIb alpha binding, lying on the surface distal to the mutation site. A similar finding has been observed for the VWF-A1 crystal structure containing the identical mutation (FIG. 4).

This altered conformation represents a high affinity binding state of the A1 domain. However, the pathway of allosteric change proposed previously, involving the burial of a water molecule, cannot be a general feature of type 2B mutants, and the structural rearrangements appear too subtle to explain the altered kinetics. Interestingly, complex formation between botrocetin and VWF-A1 in which the type 2B mutation 1309I>V has been incorporated, and has demonstrated that most of these structural differences are reversed including: (1) loss of the buried water molecule at the mutation site; (2) the peptide plan between Asp 1323 and Gly 1324 flips back to a conformation similar to that in the WT structure; and (3) the side chain of His 1326 remains in the “mutant” position, although there is some evidence from electron density of an alternative conformation similar to WT. However, this reversion in structure does not correlate with a loss in the function-enhancing activity associated with the type 2B mutation. In fact, the addition of botrocetin further augments the interaction between the mutant A1 domain and platelets in flow (42). Thus, an alternative mechanism must account for function-enhancing nature of type 2B mutations. Moreover, these subtle alterations in structure did not compare to the large conformational changes in homologous integrin I-type domains that occur on ligand binding.

Recent findings provide support that type 2B mutations may stabilize the binding of a region of GPIb alpha known as the β-hairpin to an area near the location of these altered residues, distinct from that identified by site-directed mutagenesis. Type 2B mutations have been suggested to destabilize a network of interactions observed between the bottom face of the A1 domain and its terminal peptides in the wild type A1 structure, thereby making the binding site accessible (FIG. 6).

Progress has been made in understanding the structure of this receptor-ligand pair and potential alterations in conformation that may regulate this interaction. This model is non-limiting. However, the model permits determination of the kinetic and biomechanical basis for 1) the regulation of VWF-A1 domain activity in response to hydrodynamic forces, 2) the alterations in bond kinetics that result from incorporation of type 2B mutations into the VWF-A1 domain, and 3) the susceptibility of the kinetics of the GPIb alpha-VWF-A1 bond to an applied force.

Transgenic Animals

The invention provides for transgenic non-human animals that comprise a genome contains a nucleotide sequence (such as a gene) encoding a modified form of the A1 domain of VWF. The modification can be an amino acid residue substitution at a position involved with binding to GPIb alpha or in close proximity to this region such as, but not limited to, positions 1263, 1269, 1274, 1287, 1302, 1308, 1313, 1314, 1326, 1329, 1330, 1333, 1344, 1347, 1350, 1370, 1379, 1381, 1385 1391, 1394, 1397, 1421, 1439, 1442, 1449, 1466, 1469, 1472, 1473, 1475, 1479. It can also comprise a partial or full replacement of the animal A1 domain of VWF with the A1 domain of human VWF. It can also comprise a partial or full replacement of the entire VWF gene in an animal with the human VWF gene. Such non-human animals include vertebrates such as ovines, bovines, rodents, non-human primates, porcines, caprines, equines, ruminants, lagomorphs, canines, felines, aves, and the like. In one embodiment, non-human animals are selected from the order Rodentia that includes murines (such as rats and mice). In another embodiment, mice are particularly useful.

The transgenic non-human animals of the current invention are produced by experimental manipulation of the genome of the germline of the non-human animal (such as those animals described above). These genetically engineered non-human animals may be produced by several methods well known in the art which include the introduction of a “transgene” that comprises a nucleic acid (for example, DNA such as the A1 domain of VWF) integrated into a chromosome of the somatic and/or germ line cells of a non-human animal via methods known to one skilled in the art or into an embryonal target cell. A transgenic animal is an animal whose genome has been altered by the introduction of a transgene.

The term “transgene” as used herein refers to a foreign gene that is placed into an organism by introducing the foreign gene into newly fertilized eggs, embryonic stem (ES) cells, or early embryos. The term “foreign gene” refers to any nucleic acid (for example, a gene sequence) that is introduced into the genome of an animal by experimental manipulations. These nucleic acids may include gene sequences found in that animal so long as the introduced gene contains some modification (for example, the presence of a selectable marker gene, a point mutation—such as the base pair substitution mutant that contributes to the amino acid change at amino acid residue 1326 of the A1 domain of VWF—the presence of a loxP site, and the like) relative to the naturally-occurring gene.

The term “loxP site” refers to a short (34 bp) DNA sequence that is recognized by the Cre recombinase of the E. coli bacteriophage P1. In the presence of Cre recombinase, placement of two loxP sites in the same orientation on either side of a DNA segment can result in efficient excision of the intervening DNA segment, leaving behind only a single copy of the loxP site (Sauer and Henderson (1988) Proc. Natl. Acad. Sci. USA 85:5166).

In one embodiment, the invention provides for a targeting construct or vector that comprises a selectable marker gene flanked on either side by a modified A1 domain of VWF. The modification of the A1 domain can comprise an amino acid residue substitution at a position involved with binding to GPIb alpha (such as, but not limited to, positions 1263, 1269, 1274, 1287, 1302, 1308, 1313, 1314, 1326, 1329, 1330, 1333, 1344, 1347, 1350, 1370, 1379, 1381, 1385 1391, 1394, 1397, 1421, 1439, 1442, 1449, 1466, 1469, 1472, 1473, 1475, 1479) or can be a partial or full replacement of an animal (for example, a mouse) A1 domain of VWF with the A1 domain of human VWF. The targeting vector contains the modified A1 domain of VWF gene sequence sufficient to permit the homologous recombination of the targeting vector into at least one allele of the A1 domain of the VWF gene resident in the chromosomes of the target or recipient cell (for example, ES cells). The targeting vector will usually harbor 10 to 15 kb of DNA homologous to the A1 domain of the VWF gene, wherein this 10 to 15 kb of DNA will be divided more or less equally on each side of the selectable marker gene. The targeting vector can contain more than one selectable maker gene and when multiple selectable marker genes are utilized, the targeting vector usually contains a negative selectable marker (for example, the Herpes simplex virus tk (HSV-tk) gene) and a positive selectable marker (such as G418 or the neo gene). The positive selectable marker permits the selection of recipient cells containing an integrated copy of the targeting vector and but does not enable one skilled in the art to determine whether this integration occurred at the target site or at a random site. The presence of the negative selectable marker permits the identification of recipient cells containing the targeting vector at the targeted site (for example, which has integrated by virtue of homologous recombination into the target site). Cells growing in medium that selects against the expression of the negative selectable marker represents that the cells do not contain a copy of the negative selectable marker.

Targeting vectors can also be of the replacement-type wherein the integration of a replacement-type vector results in the insertion of a selectable marker into the target gene. Replacement-type targeting vectors may be employed to disrupt a gene (such as the VWF gene or the A1 domain of the VWF gene). This can result in the generation of a null allele; for example, an allele not capable of expressing a functional protein wherein the null alleles may be generated by deleting a portion of the coding region, deleting the entire gene, introducing an insertion and/or a frameshift mutation, and the like.

A selectable marker can include a gene which encodes an enzymatic activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed. Selectable markers may be positive. A positive selectable marker is usually a dominant selectable marker wherein the genes encode an enzymatic activity that can be detected in a mammalian cell or a cell line (including ES cells). Some non-limiting examples of dominant selectable markers include the bacterial xanthine-guanine phosphoribosyl transferase gene (also referred to as the gpt gene) which confers the ability to grow in the presence of mycophenolic acid, the bacterial hygromycin G phosphotransferase (hyg) gene which confers resistance to the antibiotic hygromycin, and the bacterial aminoglycoside 3′ phosphotransferase gene (also referred to as the neo gene) which confers resistance to the drug G418 in mammalian cells. Selectable markers may also be negative. Negative selectable markers encode an enzymatic activity whose expression is toxic to the cell when grown in an appropriate selective medium. One non-limiting example of a negative selectable marker is the HSV-tk gene wherein HSV-tk expression in cells grown in the presence of gancyclovir or acyclovir is catatonic. Growth of cells in selective medium containing acyclovir or gancyclovir therefore selects against cells capable of expressing a functional HSV TK enzyme.

The ES cells suitable of the present invention utilized to generate transgenic animals can harbor introduced expression vectors (constructs), such as plasmids and the like. The expression vector constructs can be introduced via transfection, lipofection, transformation, injection, electroporation, or infection. The expression vectors can contain coding sequences, or portions thereof, encoding proteins for expression. Such expression vectors can include the required components for the transcription and translation of the inserted coding sequence. Expression vectors containing sequences encoding the produced proteins and polypeptides, as well as the appropriate transcriptional and translational control elements, can be generated using methods well known to and practiced by those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination which are described in J. Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. and in F. M. Ausubel et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y. In one embodiment, loxP expressing targeting vectors are used for transfection methods (such as pDNR-1r vector, pACD4K-C vector, and the like). In other embodiments, Cre-recombinase-expressing plasmids are also utilized (for example, crAVE cre recombinase vectors).

Introducing targeting vectors into embryonic stem (ES) cells can generate the mutant VWF-A1 transgenic animals of the present invention. ES cells are obtained by culturing pre-implantation embryos in vitro under appropriate conditions (Evans, et al. (1981) Nature 292:154-156; Bradley, et al. (1984) Nature 309:255-258; Gossler, et al. (1986) Proc. Acad. Sci. USA 83:9065-9069; and Robertson, et al. (1986) Nature 322:445-448). Using a variety of methods known to those skilled in the art, transgenes can be efficiently introduced into the ES cells via DNA transfection methods, which include (but are not limited to), protoplast or spheroplast fusion, electroporation, retrovirus-mediated transduction, calcium phosphate co-precipitation, lipofection, microinjection, and DEAE-dextran-mediated transfection. Following the introduction into the blastocoel of a blastocyst-stage embryo, transfected ES cells can thereafter colonize an embryo and contribute to the germ line of the resulting chimeric animal (see Jaenisch, (1988) Science 240:1468-1474). Assuming that the transgene provides a means for selection, the transfected ES cells may be subjected to various selection protocols to enrich for ES cells that have integrated the transgene prior to the introduction of transfected ES cells into the blastocoel. Alternatively, the polymerase chain reaction (PCR) may be used to screen for ES cells that have integrated the transgene and precludes the need for growth of the transfected ES cells under appropriate selective conditions prior to transfer into the blastocoel.

Alternative methods for the generation of transgenic animals (such as transgenic mice) containing an altered A1 domain of the VWF gene are established in the art. For example, embryonic cells at various stages of development can be used to introduce transgenes for the production of transgenic animals and different methods are used that depend on the stage of embryonic cell development. For microinjection methods, the zygote is best suited. In the mouse, the male pronucleus reaches the size of approximately 20 microns in diameter, which allows for reproducible injection of 1-2 picoliters (pl) of suspended DNA solution. A major advantage in using zygotes as a gene transfer target is that in most cases the injected DNA will be incorporated into the host genome before the first cleavage (Brinster, et al. (1985) Proc. Natl. Acad. Sci. USA 82:4438-4442). Thus, all cells of the transgenic non-human animal (such as a mouse) will carry the incorporated transgene (mutant A1 domain of the VWF gene), which can result in the efficient transmission of the transgene to the offspring of the founder since 50% of the germ cells will harbor the transgene (see U.S. Pat. No. 4,873,191).

Another method known in the art that can be used to introduce transgenes into a non-human animal is retroviral infection. The developing non-human embryo can be cultured in vitro to the blastocyst stage wherein during this time, the blastomeres can be targets for retroviral infection (Janenich (1976) Proc. Natl. Acad. Sci. USA 73:1260-1264). Enzymatic treatment to remove the zona pellucida can increase infection efficiency of the blastomeres (Hogan et al. (1986) in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Plainview, N.Y.). The viral vector system used by one skilled in the art in order to introduce the transgene is usually a replication-defective retrovirus that harbors the transgene (Jahner, D. et al. (1985) Proc. Natl. Acad. Sci. USA 82:6927-6931; Van der Putten, et al. (1985) Proc. Natl. Acad. Sci. USA 82:6148-6152). Transfection can be easily and efficiently obtained via culturing blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart, et al. (1987) EMBO J. 6:383-388). Infection can also be performed at a later stage whereby virus or virus-producing cells are injected into the blastocoele (Jahner, D. et al. (1982) Nature 298:623-628). Most of the founder non-human animals will be mosaic for the transgene since incorporation occurs only in a subset of cells that form the transgenic animal and the founder may additionally contain various retroviral insertions of the transgene at different positions in the genome that generally will segregate in the offspring. Additional methods of using retroviruses or retroviral vectors to create transgenic animals known to those skilled in the art involves microinjecting mitomycin C-treated cells or retroviral particles producing retrovirus into the perivitelline space of fertilized eggs or early embryos (see Haskell and Bowen (1995) Mol. Reprod. Dev. 40:386).

In one embodiment of the invention, non-human transgenic animals can be generated that express a modified A1 domain of the VWF sequence. The modified A1 domain can contain an amino acid residue substitution at a position involved with binding of the VWF protein to GPIb alpha (such as, but not limited to, positions 1263, 1269, 1274, 1287, 1302, 1308, 1313, 1314, 1326, 1329, 1330, 1333, 1344, 1347, 1350, 1370, 1379, 1381, 1385 1391, 1394, 1397, 1421, 1439, 1442, 1449, 1466, 1469, 1472, 1473, 1475, 1479). In some embodiments, the VWF-A1 domain can comprise a single mutation, while in other embodiments, it can comprise 2 or more mutations.

In one embodiment of the invention, the modification of the A1 domain can be a partial or full replacement of an animal (for example, a mouse) A1 domain of VWF with the A1 domain of human VWF. In other words, the A1 domain in the animal VWF is removed and replaced with the human A1 sequence. In another embodiment, the animal VWF A1 domain may be partially replaced so that some portion of the human A1 domain replaces a portion of the animal A1 domain. For example, human A1 domain sequence could comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 99%, or 100% of the animal A1 domain. The A1 domain of human VWF protein comprises SEQ ID NO: 1. In another embodiment, the animal can be a model for pre-clinical testing of compounds, wherein the animal expresses a mutant von Willebrand Factor (VWF) A1 protein containing one or more mutations, such that the binding specificity of the mutant VWF-A1 protein changes from being specific for the animal platelets to being specific for human platelets. In another embodiment, the mutation occurs in the VWF-A1 domain of a mouse. In particular embodiments, the murine mutant VWF-A1 protein comprises at least one mutation comprising 1263P>S, 1269N>D, 1274K>R, 1287M>R, 1302G>D, 1308H>R, 1313R>W, 1314I>V, 1326R>H, 1329L>I, 1330E>G, 1333A>D, 1344T>A, 1347I>V, 1350T>A, 1370G>S, 1379H>R, 1381T>A, 1385T>M 1391P>Q, 1394A>S, 1397L>F, 1421S>N, 1439L>V, 1442G>S, 1449R>Q, 1466A>P, 1469Q>L, 1472Q>H, 1473V>M, 1475H>Q, 1479S>G, or any combination thereof. In a particularly useful embodiment, the murine mutant VWF-A1 protein comprises a 1326R>H mutation, a 1314I>V mutation, or a combination thereof.

The modification of the A1 domain can be an amino acid substitution at residue 1326 (for example Arg for His in the mouse). In some embodiments, the non-human transgenic animal harbors a mutant construct wherein an amino acid residue substitution at a position involved affects binding to GPIb alpha (such as, but not limited to, positions 1263, 1269, 1274, 1287, 1302, 1308, 1313, 1314, 1326, 1329, 1330, 1333, 1344, 1347, 1350, 1370, 1379, 1381, 1385 1391, 1394, 1397, 1421, 1439, 1442, 1449, 1466, 1469, 1472, 1473, 1475, 1479). In yet other embodiments, non-human transgenic animals can successfully harbor a type 2B (Ile1309Val; 1309I>V) mutation and/or an Arg1326His (1326R>H) mutant construct. In another embodiment, the non-human transgenic animal expresses an Arg1326His (1326R>H) mutation wherein the mutant VWF-A1 domain comprises SEQ ID NO: 5, which corresponds to the His amino acid at the same position in humans, canines, chimpanzees, rat, porcine, felines, equines, bovine, and the like (Jenkins et al., (1998) Blood 91(6): 2032-44). In further embodiments of the invention, the non-human transgenic animal is a mouse. Example 3 below describes the transgenic animal of the current invention.

Molecular Manipulations of VWF-A1 and its Corresponding Mutants

The present invention utilizes conventional molecular biology, microbiology, and recombinant DNA techniques available to one of ordinary skill in the art. Such techniques are well known to the skilled worker and are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, “Molecular Cloning: A Laboratory Manual” (1982): “DNA Cloning: A Practical Approach,” Volumes I and II (D. N. Glover, ed., 1985); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Nucleic Acid Hybridization” (B. D. Hames & S. J. Higgins, eds., 1985); “Transcription and Translation” (B. D. Hames & S. J. Higgins, eds., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1986); “Immobilized Cells and Enzymes” (IRL Press, 1986): B. Perbal, “A Practical Guide to Molecular Cloning” (1984), and Sambrook, et al., “Molecular Cloning: a Laboratory Manual” (1989).

The VWF sequences from mouse, human, have been aligned as shown in Jenkins et al. (1998) “Molecular Modeling of Ligand and Mutation Sites of the Type A Domains of Human von Willebrand Factor and Their Relevance to von Willebrand's Disease” Vol. 91, No. 6, Blood, pp. 2032-2044.

The DNA and polypeptide sequences of human VWF are readily available to those skilled in the art, under Genbank Accession No. X04385. The polypeptide sequence of the A1 domain of human VWF, which runs from amino acid residue number 1260 to amino acid residue number 1480 of the nucleotide sequence of SEQ ID NO:6, is shown in SEQ ID NO: 1. The polypeptide sequence of the A1 domain of mouse VWF, which runs from amino acid residue number 1260 to amino acid residue number 1480 of the of SEQ ID NO:8, is shown in SEQ ID NO: 2.

SEQ ID NO: 1 is the human wild type amino acid sequence corresponding to the A1 domain of VWF. The residues shown in SEQ ID NO: 1 are residues 1260-1480, the A1 domain, of SEQ ID NO: 6.

SEQ ID NO:1: EDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKAFVVDMMERLRI SQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYAGSQVASTSE VLKYTLFQIFSKIDRPEASRIALLLMASQEPQRMSRNFVRYVQGLKKKKV IVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLCD LAPEAPPPTLPPHMAQVTVGP

SEQ ID NO: 2 is the mouse wild type amino acid sequence corresponding to the A1 domain of VWF. The residues shown in SEQ ID NO:2 are residue numbers 1260-1480 from the full length mouse VWF shown in SEQ ID NO: 8.

SEQ ID NO:2: EDTPEPPLHNFYCSKLLDLVFLLDGSSMLSEAEFEVLKAFVVGMMERLHI SQKRIRVAVVEYHDGSRAYLELKARKRPSELRRITSQIKYTGSQVASTSE VLKYTLFQIFGKIDRPEASHITLLLTASQEPPRMARNLVRYVQGLKKKKV IVIPVGIGPHASLKQIRLIEKQAPENKAFLLSGVDELEQRRDEIVSYLCD LAPEAPAPTQPPQVAHVTVSP

The nucleotide sequence of the A1 domain of human VWF corresponding to amino acid residues 1260-1480 is shown in SEQ ID NO: 3 and of mouse VWF is shown in SEQ ID NO: 4 below.

SEQ ID NO: 3 is the human wild type nucleotide sequence corresponding to the A1 domain of VWF:

GAGGACATCTCGGAACCGCCGTTGCACGATTTCTACTGCAGCAGGCTACT GGACCTGGTCTTCCTGCTGGATGGCTCCTCCAGGCTGTCCGAGGCTGAGT TTGAAGTGCTGAAGGCCTTTGTGGTGGACATGATGGAGCGGCTGCGCATC TCCCAGAAGTGGGTCCGCGTGGCCGTGGTGGAGTACCACGACGGCTCCCA CGCCTACATCGGGCTCAAGGACCGGAAGCGACCGTCAGAGCTGCGGCGCA TTGCCAGCCAGGTGAAGTATGCGGGCAGCCAGGTGGCCTCCACCAGCGAG GTCTTGAAATACACACTGTTCCAAATCTTCAGCAAGATCGACCGCCCTGA AGCCTCCCGCATCGCCCTGCTCCTGATGGCCAGCCAGGAGCCCCAACGGA TGTCCCGGAACTTTGTCCGCTACGTCCAGGGCCTGAAGAAGAAGAAGGTC ATTGTGATCCCGGTGGGCATTGGGCCCCATGCCAACCTCAAGCAGATCCG CCTCATCGAGAAGCAGGCCCCTGAGAACAAGGCCTTCGTGCTGAGCAGTG TGGATGAGCTGGAGCAGCAAAGGGACGAGATCGTTAGCTACCTCTGTGAC CTTGCCCCTGAAGCCCCTCCTCCTACTCTGCCCCCCCACATGGCACAAGT CACTGTGGGCCCG

SEQ ID NO: 4 is the mouse wild type nucleotide sequence corresponding to the A1 domain of VWF:

GAGGATACCCCCGAGCCCCCCCTGCACAACTTCTACTGCAGCAAGCTGCT GGATCTTGTCTTCCTGCTGGATGGCTCCTCTATGTTGTCCGAGGCTGAGT TTGAAGTGCTCAAAGCTTTTGTGGTGGGCATGATGGAGAGGTTACACATC TCTCAGAAGCGCATCCGCGTGGCAGTGGTAGAGTACCATGATGGCTCCCG TGCCTACCTTGAGCTCAAGGCCCGGAAGCGACCCTCAGAGCTTCGGCGCA TCACCAGCCAGATTAAGTATACAGGCAGCCAGGTGGCCTCTACCAGTGAG GTTTTGAAGTACACACTGTTCCAGATCTTTGGCAAAATTGACCGCCCTGA AGCCTCCCATATCACTCTGCTCCTGACTGCTAGCCAGGAGCCCCCACGGA TGGCTAGGAATTTGGTCCGCTATGTCCAAGGTCTGAAGAAGAAGAAGGTT ATCGTGATCCCTGTGGGCATTGGGCCCCACGCCAGCCTCAAACAGATCCG CCTCATCGAGAAGCAGGCCCCTGAAAACAAGGCTTTTCTGCTCAGTGGGG TGGATGAGCTGGAGCAGAGAAGAGATGAGATAGTCAGCTACCTCTGTGAC CTTGCTCCCGAGGCCCCAGCCCCAACTCAGCCTCCACAGGTAGCCCACGT CACCGTGAGTCCA

Human mRNA for pre-pro-von Willebrand factor:

SEQ ID NO: 6: Amino Acid Sequence Human VWF-(residue 1 to residue 2813)    1 MIPARFAGVL LALALILPGT LCAEGTRGRS STARCSLFGS DFVNTFDGSM YSFAGYCSYL   61 LAGGCQKRSF SIIGDFQNGK RVSLSVYLGE FFDIHLFVNG TVTQGDQRVS MPYASKGLYL  121 ETEAGYYKLS GEAYGFVARI DGSGNFQVLL SDRYFNKTCG LCGNFNIFAE DDFMTQEGTL  181 TSDPYDFANS WALSSGEQWC ERASPPSSSC NISSGEMQKG LWEQCQLLKS TSVFARCHPL  241 VDPEPFVALC EKTLCECAGG LECACPALLE YARTCAQEGM VLYGWTDHSA CSPVCPAGME  301 YRQCVSPCAR TCQSLHINEM CQERCVDGCS CPEGQLLDEG LCVESTECPC VHSGKRYPPG  361 TSLSRDCNTC ICRNSQWICS NEECPGECLV TGQSHFKSFD NRYFTFSGIC QYLLARDCQD  421 HSFSIVIETV QCADDRDAVC TRSVTVRLPG LHNSLVKLKH GAGVAMDGQD IQLPLLKGDL  481 RIQHTVTASV RLSYGEDLQM DWDGRGRLLV KLSPVYAGKT CGLCGNYNGN QGDDFLTPSG  541 LAEPRVEDFG NAWKLHGDCQ DLQKQHSDPC ALNPRMTRFS EEACAVLTSP TFEACHRAVS  601 PLPYLRNCRY DVCSCSDGRE CLCGALASYA AACAGRGVRV AWREPGRCEL NCPKGQVYLQ  661 CGTPCNLTCR SLSYPDEECN EACLEGCFCP PGLYMDERGD CVPKAQCPCY YDGEIFQPED  721 IFSDHHTMCY CEDGFMHCTM SGVPGSLLPD AVLSSPLSHR SKRSLSCRPP MVKLVCPADN  781 LRAEGLECTK TCQNYDLECM SMGCVSGCLC PPGMVRHENR CVALERCPCF HQGKEYAPGE  841 TVKIGCNTCV CRDRKWNCTD HVCDATCSTI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS  901 NPGTFRILVG NKGCSHPSVK CKKRVTILVE GGEIELFDGE VNVKRPMKDE THFEVVESGR  961 YIILLLGKAL SVVWDRHLSI SVVLKQTYQE KVCGLCGNFD GIQNNDLTSS NLQVEEDPVD 1021 FGNSWKVSSQ CADTRKVPLD SSPATCHNNI MKQTMVDSSC RILTSDVFQD CNKLVDPEPY 1081 LDVCIYDTCS CESIGDCACF CDTIAAYAHV CAQHGKVVTW RTATLCPQSC EERNLRENGY 1141 ECEWRYNSCA PACQVTCQHP EPLACPVQCV EGCHAHCPPG KILDELLQTC VDPEDCPVCE 1201 VAGRRFASGK KVTLNPSDPE HCQICHCDVV NLTCEACQEP GGLVVPPTDA PVSPTTLYVE 1261 DISEPPLHDF YCSRLLDLVF LLDGSSRLSE AEFEVLKAFV VDMMERLRIS QKWVRVAVVE 1321 YHDGSHAYIG LKDRKRPSEL RRIASQVKYA GSQVASTSEV LKYTLFQIFS KIDRPEASRI 1381 ALLLMASQEP QRMSRNFVRY VQGLKKKKVI VIPVGIGPHA NLKQIRLIEK QAPENKAFVL 1441 SSVDELEQQR DEIVSYLCDL APEAPPPTLP PHMAQVTVGP GLLGVSTLGP KRNSMVLDVA 1501 FVLEGSDKIG EADFNRSKEF MEEVIQRMDV GQDSIHVTVL QYSYMVTVEY PFSEAQSKGD 1561 ILQRVREIRY QGGNRTNTGL ALRYLSDHSF LVSQGDREQA PNLVYMVTGN PASDEIKRLP 1621 GDIQVVPIGV GPNANVQELE RIGWPNAPIL IQDFETLPRE APDLVLQRCC SGEGLQIPTL 1681 SPAPDCSQPL DVILLLDGSS SFPASYFDEM KSFAKAFISK ANIGPRLTQV SVLQYGSITT 1741 IDVPWNVVPE KAHLLSLVDV MQREGGPSQI GDALGFAVRY LTSEMHGARP GASKAVVILV 1801 TDVSVDSVDA AADAARSNRV TVFPIGIGDR YDAAQLRILA GPAGDSNVVK LQRIEDLPTM 1861 VTLGNSFLHK LCSGFVRICM DEDGNEKRPG DVWTLPDQCH TVTCQPDGQT LLKSHRVNCD 1921 RGLRPSCPNS QSPVKVEETC GCRWTCPCVC TGSSTRHIVT FDGQNFKLTG SCSYVLFQNK 1981 EQDLEVILHN GACSPGARQG CMKSIEVKHS ALSVELHSDM EVTVNGRLVS VPYVGGNMEV 2041 NVYGAIMHEV RFNHLGHIFT FTPQNNEFQL QLSPKTFASK TYGLCGICDE NGANDFMLRD 2101 GTVTTDWKTL VQEWTVQRPG QTCQPILEEQ CLVPDSSHCQ VLLLPLFAEC HKVLAPATFY 2161 AICQQDSCHQ EQVCEVIASY AHLCRTNGVC VDWRTPDFCA MSCPPSLVYN HCEHGCPRHC 2221 DGNVSSCGDH PSEGCFCPPD KVMLEGSCVP EEACTQCIGE DGVQHQFLEA WVPDHQPCQI 2281 CTCLSGRKVN CTTQPCPTAK APTCGLCEVA RLRQNADQCC PEYECVCDPV SCDLPPVPHC 2341 ERGLQPTLTN PGECRPNFTC ACRKEECKRV SPPSCPPHRL PTLRKTQCCD EYECACNCVN 2401 STVSCPLGYL ASTATNDCGC TTTTCLPDKV CVHRSTIYPV GQFWEEGCDV CTCTDMEDAV 2461 MGLRVAQCSQ KPCEDSCRSG FTYVLHEGEC CGRCLPSACE VVTGSPRGDS QSSWKSVGSQ 2521 WASPENPCLI NECVRVKEEV FIQQRNVSCP QLEVPVCPSG FQLSCKTSAC CPSCRCERME 2581 ACMLNGTVIG PGKTVMIDVC TTCRCMVQVG VISGFKLECR KTTCNPCPLG YKEENNTGEC 2641 CGRCLPTACT IQLRGGQIMT LKRDETLQDG CDTHFCKVNE RGEYFWEKRV TGCPPFDEHK 2701 CLAEGGKIMK IPGTCCDTCE EPECNDITAR LQYVKVGSCK SEVEVDIHYC QGKCASKAMY 2761 SIDINDVQDQ CSCCSPTRTE PMQVALHCTN GSVVYHEVLN AMECKCSPRK CSK VWF mature peptide (AA 763-2790) VWF pro polypeptide (AA 1-2790) SEQ ID NO: 7 Nucleic acid sequence-human VWF    1 agctcacagc tattgtggtg ggaaagggag ggtggttggt ggatgtcaca gcttgggctt   61 tatctccccc agcagtgggg actccacagc ccctgggcta cataacagca agacagtccg  121 gagctgtagc agacctgatt gagcctttgc agcagctgag agcatggcct agggtgggcg  181 gcaccattgt ccagcagctg agtttcccag ggaccttgga gatagccgca gccctcattt  241 gcaggggaag gcaccattgt ccagcagctg agtttcccag ggaccttgga gatagccgca  301 gccctcattt atgattcctg ccagatttgc cggggtgctg cttgctctgg ccctcatttt  361 gccagggacc ctttgtgcag aaggaactcg cggcaggtca tccacggccc gatgcagcct  421 tttcggaagt gacttcgtca acacctttga tgggagcatg tacagctttg cgggatactg  481 cagttacctc ctggcagggg gctgccagaa acgctccttc tcgattattg gggacttcca  541 gaatggcaag agagtgagcc tctccgtgta tcttggggaa ttttttgaca tccatttgtt  601 tgtcaatggt accgtgacac agggggacca aagagtctcc atgccctatg cctccaaagg  661 gctgtatcta gaaactgagg ctgggtacta caagctgtcc ggtgaggcct atggctttgt  721 ggccaggatc gatggcagcg gcaactttca agtcctgctg tcagacagat acttcaacaa  781 gacctgcggg ctgtgtggca actttaacat ctttgctgaa gatgacttta tgacccaaga  841 agggaccttg acctcggacc cttatgactt tgccaactca tgggctctga gcagtggaga  901 acagtggtgt gaacgggcat ctcctcccag cagctcatgc aacatctcct ctggggaaat  961 gcagaagggc ctgtgggagc agtgccagct tctgaagagc acctcggtgt ttgcccgctg 1021 ccaccctctg gtggaccccg agccttttgt ggccctgtgt gagaagactt tgtgtgagtg 1081 tgctgggggg ctggagtgcg cctgccctgc cctcctggag tacgcccgga cctgtgccca 1141 ggagggaatg gtgctgtacg gctggaccga ccacagcgcg tgcagcccag tgtgccctgc 1201 tggtatggag tataggcagt gtgtgtcccc ttgcgccagg acctgccaga gcctgcacat 1261 caatgaaatg tgtcaggagc gatgcgtgga tggctgcagc tgccctgagg gacagctcct 1321 ggatgaaggc ctctgcgtgg agagcaccga gtgtccctgc gtgcattccg gaaagcgcta 1381 ccctcccggc acctccctct ctcgagactg caacacctgc atttgccgaa acagccagtg 1441 gatctgcagc aatgaagaat gtccagggga gtgccttgtc actggtcaat cccacttcaa 1501 gagctttgac aacagatact tcaccttcag tgggatctgc cagtacctgc tggcccggga 1561 ttgccaggac cactccttct ccattgtcat tgagactgtc cagtgtgctg atgaccgcga 1621 cgctgtgtgc acccgctccg tcaccgtccg gctgcctggc ctgcacaaca gccttgtgaa 1681 actgaagcat ggggcaggag ttgccatgga tggccaggac atccagctcc ccctcctgaa 1741 aggtgacctc cgcatccagc atacagtgac ggcctccgtg cgcctcagct acggggagga 1801 cctgcagatg gactgggatg gccgcgggag gctgctggtg aagctgtccc ccgtctacgc 1861 cgggaagacc tgcggcctgt gtgggaatta caatggcaac cagggcgacg acttccttac 1921 cccctctggg ctggcagagc cccgggtgga ggacttcggg aacgcctgga agctgcacgg 1981 ggactgccag gacctgcaga agcagcacag cgatccctgc gccctcaacc cgcgcatgac 2041 caggttctcc gaggaggcgt gcgcggtcct gacgtccccc acattcgagg cctgccatcg 2101 tgccgtcagc ccgctgccct acctgcggaa ctgccgctac gacgtgtgct cctgctcgga 2161 cggccgcgag tgcctgtgcg gcgccctggc cagctatgcc gcggcctgcg cggggagagg 2221 cgtgcgcgtc gcgtggcgcg agccaggccg ctgtgagctg aactgcccga aaggccaggt 2281 gtacctgcag tgcgggaccc cctgcaacct gacctgccgc tctctctctt acccggatga 2341 ggaatgcaat gaggcctgcc tggagggctg cttctgcccc ccagggctct acatggatga 2401 gaggggggac tgcgtgccca aggcccagtg cccctgttac tatgacggtg agatcttcca 2461 gccagaagac atcttctcag accatcacac catgtgctac tgtgaggatg gcttcatgca 2521 ctgtaccatg agtggagtcc ccggaagctt gctgcctgac gctgtcctca gcagtcccct 2581 gtctcatcgc agcaaaagga gcctatcctg tcggcccccc atggtcaagc tggtgtgtcc 2641 cgctgacaac ctgcgggctg aagggctcga gtgtaccaaa acgtgccaga actatgacct 2701 ggagtgcatg agcatgggct gtgtctctgg ctgcctctgc cccccgggca tggtccggca 2761 tgagaacaga tgtgtggccc tggaaaggtg tccctgcttc catcagggca aggagtatgc 2821 ccctggagaa acagtgaaga ttggctgcaa cacttgtgtc tgtcgggacc ggaagtggaa 2881 ctgcacagac catgtgtgtg atgccacgtg ctccacgatc ggcatggccc actacctcac 2941 cttcgacggg ctcaaatacc tgttccccgg ggagtgccag tacgttctgg tgcaggatta 3001 ctgcggcagt aaccctggga cctttcggat cctagtgggg aataagggat gcagccaccc 3061 ctcagtgaaa tgcaagaaac gggtcaccat cctggtggag ggaggagaga ttgagctgtt 3121 tgacggggag gtgaatgtga agaggcccat gaaggatgag actcactttg aggtggtgga 3181 gtctggccgg tacatcattc tgctgctggg caaagccctc tccgtggtct gggaccgcca 3241 cctgagcatc tccgtggtcc tgaagcagac ataccaggag aaagtgtgtg gcctgtgtgg 3301 gaattttgat ggcatccaga acaatgacct caccagcagc aacctccaag tggaggaaga 3361 ccctgtggac tttgggaact cctggaaagt gagctcgcag tgtgctgaca ccagaaaagt 3421 gcctctggac tcatcccctg ccacctgcca taacaacatc atgaagcaga cgatggtgga 3481 ttcctcctgt agaatcctta ccagtgacgt cttccaggac tgcaacaagc tggtggaccc 3541 cgagccatat ctggatgtct gcatttacga cacctgctcc tgtgagtcca ttggggactg 3601 cgcctgcttc tgcgacacca ttgctgccta tgcccacgtg tgtgcccagc atggcaaggt 3661 ggtgacctgg aggacggcca cattgtgccc ccagagctgc gaggagagga atctccggga 3721 gaacgggtat gagtgtgagt ggcgctataa cagctgtgca cctgcctgtc aagtcacgtg 3781 tcagcaccct gagccactgg cctgccctgt gcagtgtgtg gagggctgcc atgcccactg 3841 ccctccaggg aaaatcctgg atgagctttt gcagacctgc gttgaccctg aagactgtcc 3901 agtgtgtgag gtggctggcc ggcgttttgc ctcaggaaag aaagtcacct tgaatcccag 3961 tgaccctgag cactgccaga tttgccactg tgatgttgtc aacctcacct gtgaagcctg 4021 ccaggagccg ggaggcctgg tggtgcctcc cacagatgcc ccggtgagcc ccaccactct 4081 gtatgtggag gacatctcgg aaccgccgtt gcacgatttc tactgcagca ggctactgga 4141 cctggtcttc ctgctggatg gctcctccag gctgtccgag gctgagtttg aagtgctgaa 4201 ggcctttgtg gtggacatga tggagcggct gcgcatctcc cagaagtggg tccgcgtggc 4261 cgtggtggag taccacgacg gctcccacgc ctacatcggg ctcaaggacc ggaagcgacc 4321 gtcagagctg cggcgcattg ccagccaggt gaagtatgcg ggcagccagg tggcctccac 4381 cagcgaggtc ttgaaataca cactgttcca aatcttcagc aagatcgacc gccctgaagc 4441 ctcccgcatc gccctgctcc tgatggccag ccaggagccc caacggatgt cccggaactt 4501 tgtccgctac gtccagggcc tgaagaagaa gaaggtcatt gtgatcccgg tgggcattgg 4561 gccccatgcc aacctcaagc agatccgcct catcgagaag caggcccctg agaacaaggc 4621 cttcgtgctg agcagtgtgg atgagctgga gcagcaaagg gacgagatcg ttagctacct 4681 ctgtgacctt gcccctgaag cccctcctcc tactctgccc ccccacatgg cacaagtcac 4741 tgtgggcccg gggctcttgg gggtttcgac cctggggccc aagaggaact ccatggttct 4801 ggatgtggcg ttcgtcctgg aaggatcgga caaaattggt gaagccgact tcaacaggag 4861 caaggagttc atggaggagg tgattcagcg gatggatgtg ggccaggaca gcatccacgt 4921 cacggtgctg cagtactcct acatggtgac cgtggagtac cccttcagcg aggcacagtc 4981 caaaggggac atcctgcagc gggtgcgaga gatccgctac cagggcggca acaggaccaa 5041 cactgggctg gccctgcggt acctctctga ccacagcttc ttggtcagcc agggtgaccg 5101 ggagcaggcg cccaacctgg tctacatggt caccggaaat cctgcctctg atgagatcaa 5161 gaggctgcct ggagacatcc aggtggtgcc cattggagtg ggccctaatg ccaacgtgca 5221 ggagctggag aggattggct ggcccaatgc ccctatcctc atccaggact ttgagacgct 5281 cccccgagag gctcctgacc tggtgctgca gaggtgctgc tccggagagg ggctgcagat 5341 ccccaccctc tcccctgcac ctgactgcag ccagcccctg gacgtgatcc ttctcctgga 5401 tggctcctcc agtttcccag cttcttattt tgatgaaatg aagagtttcg ccaaggcttt 5461 catttcaaaa gccaatatag ggcctcgtct cactcaggtg tcagtgctgc agtatggaag 5521 catcaccacc attgacgtgc catggaacgt ggtcccggag aaagcccatt tgctgagcct 5581 tgtggacgtc atgcagcggg agggaggccc cagccaaatc ggggatgcct tgggctttgc 5641 tgtgcgatac ttgacttcag aaatgcatgg tgccaggccg ggagcctcaa aggcggtggt 5701 catcctggtc acggacgtct ctgtggattc agtggatgca gcagctgatg ccgccaggtc 5761 caacagagtg acagtgttcc ctattggaat tggagatcgc tacgatgcag cccagctacg 5821 gatcttggca ggcccagcag gcgactccaa cgtggtgaag ctccagcgaa tcgaagacct 5881 ccctaccatg gtcaccttgg gcaattcctt cctccacaaa ctgtgctctg gatttgttag 5941 gatttgcatg gatgaggatg ggaatgagaa gaggcccggg gacgtctgga ccttgccaga 6001 ccagtgccac accgtgactt gccagccaga tggccagacc ttgctgaaga gtcatcgggt 6061 caactgtgac cgggggctga ggccttcgtg ccctaacagc cagtcccctg ttaaagtgga 6121 agagacctgt ggctgccgct ggacctgccc ctgcgtgtgc acaggcagct ccactcggca 6181 catcgtgacc tttgatgggc agaatttcaa gctgactggc agctgttctt atgtcctatt 6241 tcaaaacaag gagcaggacc tggaggtgat tctccataat ggtgcctgca gccctggagc 6301 aaggcagggc tgcatgaaat ccatcgaggt gaagcacagt gccctctccg tcgagctgca 6361 cagtgacatg gaggtgacgg tgaatgggag actggtctct gttccttacg tgggtgggaa 6421 catggaagtc aacgtttatg gtgccatcat gcatgaggtc agattcaatc accttggtca 6481 catcttcaca ttcactccac aaaacaatga gttccaactg cagctcagcc ccaagacttt 6541 tgcttcaaag acgtatggtc tgtgtgggat ctgtgatgag aacggagcca atgacttcat 6601 gctgagggat ggcacagtca ccacagactg gaaaacactt gttcaggaat ggactgtgca 6661 gcggccaggg cagacgtgcc agcccatcct ggaggagcag tgtcttgtcc ccgacagctc 6721 ccactgccag gtcctcctct taccactgtt tgctgaatgc cacaaggtcc tggctccagc 6781 cacattctat gccatctgcc agcaggacag ttgccaccag gagcaagtgt gtgaggtgat 6841 cgcctcttat gcccacctct gtcggaccaa cggggtctgc gttgactgga ggacacctga 6901 tttctgtgct atgtcatgcc caccatctct ggtctacaac cactgtgagc atggctgtcc 6961 ccggcactgt gatggcaacg tgagctcctg tggggaccat ccctccgaag gctgtttctg 7021 ccctccagat aaagtcatgt tggaaggcag ctgtgtccct gaagaggcct gcactcagtg 7081 cattggtgag gatggagtcc agcaccagtt cctggaagcc tgggtcccgg accaccagcc 7141 ctgtcagatc tgcacatgcc tcagcgggcg gaaggtcaac tgcacaacgc agccctgccc 7201 cacggccaaa gctcccacgt gtggcctgtg tgaagtagcc cgcctccgcc agaatgcaga 7261 ccagtgctgc cccgagtatg agtgtgtgtg tgacccagtg agctgtgacc tgcccccagt 7321 gcctcactgt gaacgtggcc tccagcccac actgaccaac cctggcgagt gcagacccaa 7381 cttcacctgc gcctgcagga aggaggagtg caaaagagtg tccccaccct cctgcccccc 7441 gcaccgtttg cccacccttc ggaagaccca gtgctgtgat gagtatgagt gtgcctgcaa 7501 ctgtgtcaac tccacagtga gctgtcccct tgggtacttg gcctcaaccg ccaccaatga 7561 ctgtggctgt accacaacca cctgccttcc cgacaaggtg tgtgtccacc gaagcaccat 7621 ctaccctgtg ggccagttct gggaggaggg ctgcgatgtg tgcacctgca ccgacatgga 7681 ggatgccgtg atgggcctcc gcgtggccca gtgctcccag aagccctgtg aggacagctg 7741 tcggtcgggc ttcacttacg ttctgcatga aggcgagtgc tgtggaaggt gcctgccatc 7801 tgcctgtgag gtggtgactg gctcaccgcg gggggactcc cagtcttcct ggaagagtgt 7861 cggctcccag tgggcctccc cggagaaccc ctgcctcatc aatgagtgtg tccgagtgaa 7921 ggaggaggtc tttatacaac aaaggaacgt ctcctgcccc cagctggagg tccctgtctg 7981 cccctcgggc tttcagctga gctgtaagac ctcagcgtgc tgcccaagct gtcgctgtga 8041 gcgcatggag gcctgcatgc tcaatggcac tgtcattggg cccgggaaga ctgtgatgat 8101 cgatgtgtgc acgacctgcc gctgcatggt gcaggtgggg gtcatctctg gattcaagct 8161 ggagtgcagg aagaccacct gcaacccctg ccccctgggt tacaaggaag aaaataacac 8221 aggtgaatgt tgtgggagat gtttgcctac ggcttgcacc attcagctaa gaggaggaca 8281 gatcatgaca ctgaagcgtg atgagacgct ccaggatggc tgtgatactc acttctgcaa 8341 ggtcaatgag agaggagagt acttctggga gaagagggtc acaggctgcc caccctttga 8401 tgaacacaag tgtctggctg agggaggtaa aattatgaaa attccaggca cctgctgtga 8461 cacatgtgag gagcctgagt gcaacgacat cactgccagg ctgcagtatg tcaaggtggg 8521 aagctgtaag tctgaagtag aggtggatat ccactactgc cagggcaaat gtgccagcaa 8581 agccatgtac tccattgaca tcaacgatgt gcaggaccag tgctcctgct gctctccgac 8641 acggacggag cccatgcagg tggccctgca ctgcaccaat ggctctgttg tgtaccatga 8701 ggttctcaat gccatggagt gcaaatgctc ccccaggaag tgcagcaagt gaggctgctg 8761 cagctgcatg ggtgcctgct gctgcctgcc ttggcctgat ggccaggcca gagtgctgcc 8821 agtcctctgc atgttctgct cttgtgccct tctgagccca caataaaggc tgagctctta 8881 tcttgctgca tgttctgctc ttgtgccctt ctgagcccac aat Mus musculus strain CASA/RkJ VWF (Vwf) mRNA SEQ ID NO: 8-Amino Acid Sequence of Mouse VWF (residue no. 1 to residue no. 2813)    1 MNPFRYEICL LVLALTWPGT LCTEKPRDRP STARCSLFGD DFINTFDETM YSFAGGCSYL   61 LAGDCQKRSF SILGNFQDGK RMSLSVYLGE FFDIHLFANG TVTQGDQSIS MPYASQGLYL  121 EREAGYYKLS SETFGFAARI DGNGNFQVLM SDRHFNKTCG LCGDFNIFAE DDFRTQEGTL  181 TSDPYDFANS WALSSEEQRC KRASPPSRNC ESSSGDMHQA MWEQCQLLKT ASVFARCHPL  241 VDPESFVALC EKILCTCATG PECACPVLLE YARTCAQEGM VLYGWTDHSA CRPACPAGME  301 YKECVSPCPR TCQSLSINEV CQQQCVDGCS CPEGELLDED RCVQSSDCPC VHAGKRYPPG  361 TSLSQDCNTC ICRNSLWICS NEECPGECLV TGQSHFKSFD NRYFTFSGIC QYLLARDCED  421 HTFSIVIETM QCADDPDAVC TRSVSVRLSA LHNSLVKLKH GGAVGIDGQD VQLPFLQGDL  481 RIQHTVMASV RLSYAEDLQM DWDGRGRLLV KLSPVYSGKT CGLCGNYNGN KGDDFLTPAG  541 LVEPLVVDFG NAWKLQGDCS DLRRQHSDPC SLNPRLTRFA EEACALLTSS KFEACHHAVS  601 PLPYLQNCRY DVCSCSDSRD CLCNAVANYA AECARKGVHI GWREPGFCAL GCPQGQVYLQ  661 CGNSCNLTCR SLSLPDEECS EVCLEGCYCP PGLYQDERGD CVPKAQCPCY YDGELFQPAD  721 IFSDHHTMCY CEDGFMHCTT SGTLGSLLPD TVLSSPLSHR SKRSLSCRPP MVKLVCPADN  781 PRAQGLECAK TCQNYDLECM SLGCVSGCLC PPGMVRHENK CVALERCPCF HQGAEYAPGD  841 TVKIGCNTCV CRERKWNCTN HVCDATCSAI GMAHYLTFDG LKYLFPGECQ YVLVQDYCGS  901 NPGTFQILVG NEGCSYPSVK CRKRVTILVD GGELELFDGE VNVKRPLRDE SHFEVVESGR  961 YVILLLGQAL SVVWDHHLSI SVVLKHTYQE QVCGLCGNFD GIQNNDFTTS SLQVEEDPVN 1021 FGNSWKVSSQ CADTRKLSLD VSPATCHNNI MKQTMVDSAC RILTSDVFQG CNRLVDPEPY 1081 LDICIYDTCS CESIGDCACF CDTIAAYAHV CAQHGQVVAW RTPTLCPQSC EEKNVRENGY 1141 ECEWRYNSCA PACPVTCQHP EPLACPVQCV EGCHAHCPPG RILDELLQTC VDPQDCPVCE 1201 VAGRRLAPGK KITLSPDDPA HCQNCHCDGV NLTCEACQEP GGLVAPPTDA PVSSTTPYVE 1261 DTPEPPLHNF YCSKLLDLVF LLDGSSMLSE AEFEVLKAFV VGMMERLHIS QKRIRVAVVE 1321 YHDGSRAYLE LKARKRPSEL RRITSQIKYT GSQVASTSEV LKYTLFQIFG KIDRPEASHI 1381 TLLLTASQEP PRMARNLVRY VQGLKKKKVI VIPVGIGPHA SLKQIRLIEK QAPENKAFLL 1441 SGVDELEQRR DETVSYLCDL APEAPAPTQP PQVAHVTVSP GIAGISSPGP KRKSMVLDVV 1501 FVLEGSDEVG EANFNKSKEF VEEVIQRMDV SPDATRISVL QYSYTVTMEY AFNGAQSKEE 1561 VLRHVREIRY QGGNRTNTGQ ALQYLSEHSF SPSQGDRVEA PNLVYMVTGN PASDEIKRLP 1621 GDIQVVPIGV GPHANMQELE RISRPIAPIF IRDFETLPRE APDLVLQTCC SKEGLQLPTL 1681 PPLPDCSQPL DVVLLLDGSS SLPESSFDKM KSFAKAFISK ANIGPHLTQV SVIQYGSINT 1741 IDVPWNVVQE KAHLQSLVDL MQQEGGPSQI GDALAFAVRY VTSQIHGARP GASKAVVIII 1801 MDTSLDPVDT AADAARSNRV AVFPVGVGDR YDEAQLRILA GPGASSNVVK LQQVEDLSTM 1861 ATLGNSFFHK LCSGFSGVCV DEDGNEKRPG DVWTLPDQCH TVTCLANGQT LLQSHRVNCD 1921 HGPRPSCANS QSPVRVEETC GCRWTCPCVC TGSSTRHIVT FDGQNFKLTG SCSYVIFQNK 1981 EQDLEVLLHN GACSPGAKQA CMKSIEIKHA GVSAELHSNM EMAVDGRLVL APYVGENMEV 2041 SIYGAIMYEV RFTHLGHILT YTPQNNEFQL QLSPKTFASK MHGLCGICDE NGANDFTLRD 2101 GTVTTDWKRL VQEWTVQQPG YTCQAVPEEQ CPVSDSSHCQ VLLSASFAEC HKVIAPATFH 2161 TICQQDSCHQ ERVCEVIASY AHLCRTSGVC VDWRTTDFCA MSCPPSLVYN HCERGCPRHC 2221 DGNTSFCGDH PSEGCFCPQH QVFLEGSCVP EEACTQCVGE DGVRHQFLET WVPDHQPCQI 2281 CMCLSGRKIN CTAQPCPTAR APTCGPCEVA RLKQSTNLCC PEYECVCDLF NCNLPPVPPC 2341 EGGLQPTLTN PGECRPTFTC ACRKEECKRV SPPSCPPHRT PTLRKTQCCD EYECACSCVN 2401 STLSCPLGYL ASATTNDCGC TTTTCLPDKV CVHRGTVYPV GQFWEEGCDT CTCTDMEDTV 2461 VGLRVVQCSQ RPCEDSCQPG FSYVLHEGEC CGRCLPSACK VVAGSLRGDS HSSWKSVGSR 2521 WAVPENPCLV NECVRVEDAV FVQQRNISCP QLAVPTCPTG FQLNCETSEC CPSCHCEPVE 2581 ACLLNGTIIG PGKSVMVDLC TTCRCIVQTD AISRFKLECR KTTCEACPMG YREEKSQGEC 2641 CGRCLPTACT IQLRGGRIMT LKQDETFQDG CDSHLCRVNE RGEYIWEKRV TGCPPFDEHK 2701 CLAEGGKIVK IPGTCCDTCE EPDCKDITAK VQYIKVGDCK SQEEVDIHYC QGKCASKAVY 2761 SIDIEDVQEQ CSCCLPSRTE PMRVPLHCTN GSVVYHEVIN AMQCRCSPRN CSK SEQ ID NO: 9-Nucleic Acid Sequence of Mouse VWF    1 agtagcggct gggtttcctc aagggacctt ggagatacag cccctgtttg tatgggcaag   61 atgaaccctt tcaggtatga gatctgcctg cttgttctgg ccctcacctg gccagggacc  121 ctctgcacag aaaagccccg tgacaggccg tcgacggccc gatgcagcct ctttggggac  181 gacttcatca acacgtttga tgagaccatg tacagctttg cagggggctg cagttatctc  241 ctggctgggg actgccagaa acgttccttc tccattctcg ggaacttcca agatggcaag  301 agaatgagcc tgtctgtgta tcttggggag ttttttgaca tccatttgtt tgccaatggc  361 accgtaacgc agggtgacca aagcatctcc atgccctacg cctcccaagg actctaccta  421 gaacgcgagg ctgggtacta taagctctcc agtgagacct ttggctttgc ggccagaatc  481 gatggcaatg gcaacttcca agtcctgatg tcagacagac acttcaacaa gacctgtggg  541 ctgtgcggtg attttaacat cttcgcggaa gatgatttta ggacgcagga ggggaccttg  601 acctcagacc cctatgattt tgccaactcc tgggccctga gcagtgagga acagcggtgt  661 aaacgggcat ctcctcccag caggaactgc gagagctctt ctggggacat gcatcaggcc  721 atgtgggagc aatgccagct actgaagacg gcatcggtgt ttgcccgctg ccaccctctg  781 gtggatcccg agtcctttgt ggctctgtgt gagaagattt tgtgtacgtg tgctacgggg  841 ccagagtgcg catgtcctgt actccttgag tatgcccgaa cctgcgccca ggaagggatg  901 gtgctgtacg gctggactga ccacagtgcc tgtcgtccag cttgcccagc tggcatggaa  961 tataaggagt gtgtgtctcc ttgccccaga acctgccaga gcctgtctat caatgaagtg 1021 tgtcagcagc aatgtgtaga cggctgtagc tgccctgagg gagagctctt ggatgaagac 1081 cgatgtgtgc agagctccga ctgtccttgc gtgcacgctg ggaagcggta ccctcctggc 1141 acctccctct ctcaggactg caacacttgt atctgcagaa acagcctatg gatctgcagc 1201 aatgaggaat gcccagggga gtgtcttgtc acaggccaat cgcacttcaa gagcttcgac 1261 aacaggtact tcaccttcag tgggatctgc caatatctgc tggcccggga ctgcgaggat 1321 cacactttct ccattgtcat agagaccatg cagtgtgccg atgaccctga tgctgtctgc 1381 acccgctcgg tcagtgtgcg gctctctgcc ctgcacaaca gcctggtgaa actgaagcac 1441 gggggagcag tgggcatcga tggtcaggat gtccagctcc ccttcctgca aggtgacctc 1501 cgcatccagc acacagtgat ggcttctgta cgcctcagct atgcggagga cctgcagatg 1561 gactgggatg gccgtgggcg gctactggtt aagctgtccc cagtctattc tgggaagacc 1621 tgtggcttgt gtgggaatta caacggcaac aagggagacg acttcctcac gccggccggc 1681 ttggtggagc ccctggtggt agacttcgga aacgcctgga agcttcaagg ggactgttcg 1741 gacctgcgca ggcaacacag cgacccctgc agcctgaatc cacgcttgac caggtttgca 1801 gaggaggctt gtgcgctcct gacgtcctcc aagttcgagg cctgccacca cgcagtcagc 1861 cctctgccct atctgcagaa ctgccgttat gatgtttgct cctgctccga cagccgggat 1921 tgcctgtgta acgcagtagc taactatgct gccgagtgtg cccgaaaagg cgtgcacatc 1981 gggtggcggg agcctggctt ctgtgctctg ggctgtccac agggccaggt gtacctgcag 2041 tgtgggaatt cctgcaacct gacctgccgc tccctctccc tcccggatga agaatgcagt 2101 gaagtctgtc ttgaaggctg ctactgccca ccagggctct accaggatga aagaggggac 2161 tgtgtgccca aggcccagtg cccctgctac tacgatggtg agctcttcca gcctgcggac 2221 attttctcag accaccatac catgtgttac tgtgaagatg gcttcatgca ctgtaccaca 2281 agtggcaccc tggggagcct gttgcctgac actgtcctca gcagtcccct gtctcaccgt 2341 agcaaaagga gcctttcctg ccggccaccc atggtcaagc tggtgtgtcc tgctgacaac 2401 ccacgggctc aagggctgga gtgtgctaag acgtgccaga actacgacct ggagtgtatg 2461 agcctgggct gtgtgtctgg ctgcctctgt cccccaggca tggtccggca cgaaaacaag 2521 tgtgtggcct tggagcggtg tccctgcttc catcagggtg cagagtacgc cccgggagac 2581 acagtgaaga ttggctgcaa cacctgtgtc tgccgggagc ggaagtggaa ctgcacgaac 2641 catgtgtgtg acgccacttg ctctgccatt ggtatggccc actacctcac cttcgatgga 2701 ctcaagtacc tgttcccggg ggagtgccag tatgttctgg tgcaggatta ctgtggcagt 2761 aaccctggga cctttcagat cctggtggga aatgagggtt gcagctatcc ctcggtgaag 2821 tgcaggaagc gggtgaccat cctggtggat ggaggggagc ttgaactgtt tgacggagag 2881 gtgaacgtta agaggcccct gagagatgaa tctcactttg aggtggtgga gtcgggccgg 2941 tacgtcatcc tgctgctggg tcaggccctt tctgtggtct gggaccacca cctcagcatc 3001 tctgtggtcc tgaagcacac ataccaggaa caggtgtgtg gcctctgcgg gaactttgat 3061 ggcatccaga acaatgactt caccactagc agcctccagg tggaggaaga ccccgtcaac 3121 tttgggaact cctggaaagt gagctcacag tgtgctgaca cgagaaagct gtcactagat 3181 gtttcccctg ccacttgcca caacaacatc atgaaacaga cgatggtgga ctcagcctgc 3241 agaatcctta ccagtgacgt cttccagggc tgcaacaggc tggtggaccc tgagccatac 3301 ctggacatct gtatttatga cacttgctcc tgtgagtcca tcggggactg cgcctgtttc 3361 tgtgacacca ttgctgccta tgcccacgtg tgtgcccagc atggccaggt ggtagcctgg 3421 aggacaccca cactgtgccc ccagagctgt gaagaaaaga atgttcggga aaatggctat 3481 gagtgtgagt ggcgttataa cagctgtgcg cctgcttgcc cagtcacgtg tcagcaccct 3541 gagcctctgg cttgccctgt gcagtgtgtg gagggttgtc atgcacattg ccctccaggg 3601 agaatcctgg atgaacttct gcagacctgc gtagaccccc aagactgccc cgtgtgtgag 3661 gtggctggtc ggcgcttggc tcctggaaag aaaatcacct tgagtcctga tgaccctgca 3721 cactgtcaga attgtcactg tgatggtgtg aaccttacgt gtgaagcctg ccaagagccc 3781 ggaggcctgg tggcaccccc aactgatgcc ccagtcagct ctaccacccc atatgttgag 3841 gatacccccg agccccccct gcacaacttc tactgcagca agctgctgga tcttgtcttc 3901 ctgctggatg gctcctctat gttgtccgag gctgagtttg aagtgctcaa agcttttgtg 3961 gtgggcatga tggagaggtt acacatctct cagaagcgca tccgcgtggc agtggtagag 4021 taccatgatg gctcccgtgc ctaccttgag ctcaaggccc ggaagcgacc ctcagagctt 4081 cggcgcatca ccagccagat taagtataca ggcagccagg tggcctctac cagtgaggtt 4141 ttgaagtaca cactgttcca gatctttggc aaaattgacc gccctgaagc ctcccatatc 4201 actctgctcc tgactgctag ccaggagccc ccacggatgg ctaggaattt ggtccgctat 4261 gtccaaggtc tgaagaagaa gaaggttatc gtgatccctg tgggcattgg gccccacgcc 4321 agcctcaaac agatccgcct catcgagaag caggcccctg aaaacaaggc ttttctgctc 4381 agtggggtgg atgagctgga gcagagaaga gatgagatag tcagctacct ctgtgacctt 4441 gctcccgagg ccccagcccc aactcagcct ccacaggtag cccacgtcac cgtgagtcca 4501 gggatcgctg ggatctcgtc accgggacca aaacggaagt ccatggttct ggatgtggtg 4561 tttgtcctgg aggggtcaga cgaagttggt gaagccaact tcaataagag caaggagttc 4621 gtggaggagg taatccagcg catggacgtg agcccggatg caacgcgcat ctcagtactg 4681 cagtattcct acacggtaac catggagtat gccttcaatg gggcccagtc caaggaggag 4741 gtgctgcggc acgtgcgaga gatccgctac cagggcggca ataggacaaa cactgggcag 4801 gccctgcagt acctttctga gcacagcttc tctcccagcc aaggggaccg ggtagaggca 4861 cctaacctgg tctacatggt cacggggaac cccgcctctg atgagatcaa gaggttgcct 4921 ggagacatcc aggtggtacc cattggggtg ggcccccatg ccaacatgca ggaactggag 4981 aggatcagca ggcccatcgc tcccatcttc atccgggact ttgagacact tccccgagag 5041 gctcctgacc tggtcctgca gacatgttgc tccaaggagg gtctgcaact gcccaccctc 5101 ccccctctcc ctgactgcag ccaacccctg gatgtggtcc tgctcctgga tggctcctct 5161 agcttgccag agtcttcctt tgataaaatg aagagttttg ccaaggcttt catttcaaag 5221 gccaacattg ggccccacct cacacaggtg tccgtgatac agtatggaag catcaatacc 5281 attgatgtac catggaatgt ggttcaggag aaagcccatc tacagagttt ggtggacctc 5341 atgcagcagg agggtggccc cagccagatt ggggatgctc tggcctttgc cgtgcgctat 5401 gtaacttcac aaatccacgg agccaggcct ggggcctcca aagcagtggt catcatcatc 5461 atggatacct ccttggatcc cgtggacaca gcagcagatg ctgccagatc caaccgagtg 5521 gcagtgtttc ccgttggggt tggggatcgg tatgatgaag cccagctgag gatcttggca 5581 ggccctgggg ccagctccaa tgtggtaaag ctccagcaag ttgaagacct ctccaccatg 5641 gccaccctgg gcaactcctt cttccacaaa ctgtgttctg ggttttctgg agtttgtgtg 5701 gatgaagatg ggaatgagaa gaggcctggg gatgtctgga ccttgccgga tcagtgccac 5761 acagtgactt gcttggcaaa tggccagacc ttgctgcaga gtcatcgtgt caattgtgac 5821 catggacccc ggccttcatg tgccaacagc cagtctcctg ttcgggtgga ggagacgtgt 5881 ggctgccgct ggacctgccc ttgtgtgtgc acgggcagtt ccactcggca catcgtcacc 5941 ttcgatgggc agaatttcaa gcttactggt agctgctcct atgtcatctt tcaaaacaag 6001 gagcaggacc tggaagtgct cctccacaat ggggcctgca gccccggggc aaaacaagcc 6061 tgcatgaagt ccattgagat taagcatgct ggcgtctctg ctgagctgca cagtaacatg 6121 gagatggcag tggatgggag actggtcctt gccccgtacg ttggtgaaaa catggaagtc 6181 agcatctacg gcgctatcat gtatgaagtc aggtttaccc atcttggcca catcctcaca 6241 tacacgccac aaaacaacga gttccaactg cagcttagcc ccaagacctt tgcttcgaag 6301 atgcatggtc tttgcggaat ctgtgatgaa aacggggcca atgacttcac gttgcgagat 6361 ggcacggtca ccacagactg gaaaaggctt gtccaggaat ggacggtgca gcagccaggg 6421 tacacatgcc aggctgttcc cgaggagcag tgtcccgtct ctgacagctc ccactgccag 6481 gtcctcctct cagcgtcgtt tgctgaatgc cacaaggtca tcgctccagc cacattccat 6541 accatctgcc agcaagacag ttgccaccag gagcgagtgt gtgaggtgat tgcttcttac 6601 gcccatctct gtcggaccag tggggtctgt gttgattgga ggacaactga tttctgtgct 6661 atgtcatgcc caccgtccct ggtgtataac cactgtgagc gtggctgccc tcggcactgc 6721 gatgggaaca ctagcttctg tggggaccat ccctcagaag gctgcttctg tccccaacac 6781 caagtttttc tggaaggcag ctgtgtcccc gaggaggcct gcactcagtg tgttggcgag 6841 gatggagttc gacatcagtt cctggagacc tgggtcccag accatcagcc ctgtcagatc 6901 tgtatgtgcc tcagtgggag aaagattaac tgcactgccc agccgtgtcc cacagcccga 6961 gctcccacgt gtggcccatg tgaagtggct cgcctcaagc agagcacaaa cctgtgctgc 7021 ccagagtatg agtgtgtgtg tgacctgttc aactgcaact tgcctccagt gcctccgtgt 7081 gaaggagggc tccagccaac cctgaccaac cctggagaat gcagacccac ctttacctgt 7141 gcctgcagga aagaagagtg caaaagagtg tccccaccct cctgcccccc tcaccggaca 7201 cccactctcc ggaagaccca gtgctgtgat gaatacgagt gtgcttgcag ctgtgtcaac 7261 tccacgctga gctgcccact tggctacctg gcctcagcca ctaccaatga ctgtggctgc 7321 accacgacca cctgtctccc tgacaaggtt tgtgtccacc gaggcaccgt ctaccctgtg 7381 ggccagttct gggaggaggg ctgtgacacg tgcacctgta cggacatgga ggatactgtc 7441 gtgggcctgc gtgtggtcca gtgctctcaa aggccctgtg aagacagctg tcagccaggt 7501 ttttcttatg ttctccacga aggcgagtgc tgtggaaggt gcctgccctc tgcttgcaag 7561 gtggtggctg gctcactgcg gggcgattcc cactcttcct ggaaaagtgt tggatctcgg 7621 tgggctgttc ctgagaaccc ctgcctcgtc aacgagtgtg tccgcgtgga ggatgcagtg 7681 tttgtgcagc agaggaacat ctcctgccca cagctggctg tccctacctg tcccacaggc 7741 ttccaactga actgtgagac ctcagagtgc tgtcctagct gccactgtga gcctgtggag 7801 gcctgcctgc tcaatggcac catcattggg cccgggaaga gtgtgatggt tgacctatgc 7861 acgacctgcc gctgcatcgt gcagacagac gccatctcca gattcaagct ggagtgcagg 7921 aagactacct gtgaggcctg ccccatgggc tatcgggaag agaagagcca gggtgaatgc 7981 tgtgggagat gcttgcctac agcttgcact attcagctaa gaggaggacg gatcatgacc 8041 ctgaagcaag atgagacatt ccaggatggc tgtgacagtc atttgtgcag ggtcaacgag 8101 agaggagagt acatctggga gaagagggtc acgggctgcc caccatttga tgaacacaag 8161 tgtctggctg aaggaggcaa aatcgtgaaa attccaggca cctgctgtga cacatgtgag 8221 gagcctgatt gcaaagacat cacagccaag gtgcagtaca tcaaagtggg agattgtaag 8281 tcccaagagg aagtggacat tcattactgc cagggaaagt gtgccagcaa agctgtgtac 8341 tccattgaca tcgaggatgt gcaggagcaa tgctcctgct gcctgccctc gaggacggag 8401 cccatgcgcg tgcccttgca ctgcaccaat ggctctgtcg tgtaccacga ggtcatcaac 8461 gccatgcagt gcaggtgttc tccccggaac tgcagcaagt gaggcctgtg cagctacagc 8521 ggattcctac tgatacc

DNA sequences or oligonucleotides having specific sequences can be synthesized chemically or isolated by one of several approaches established in art. The basic strategies for identifying, amplifying, and isolated desired DNA sequences as well as assembling them into larger DNA molecules containing the desired sequence domains in the desired order, are well known to those of ordinary skill in the art. See, e.g., Sambrook, et al., (1989) Nature November 16; 342(6247):224-5; Perbal, B. et al., (1983) J Virol. March; 45(3):925-40. DNA segments corresponding to all or a portion of the VWF sequence may be isolated individually using the polymerase chain reaction (M. A. Innis, et al., “PCR Protocols: A Guide To Methods and Applications,” Academic Press, 1990). A complete sequence may be assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981), Nature, 292:756; Nambiar, et al. (1984), Science, 223:1299; Jay et al. (1984), J. Biol. Chem., 259:6311.

The assembled nucleotide sequence can be cloned into a suitable vector or replicon and maintained in said carrier in a composition that is substantially free of vectors that do not contain the assembled sequence, thus providing a reservoir of the assembled sequence wherein the entire sequence can be extracted from the reservoir via excising it from DNA material with restriction enzymes or by PCR amplification. Those of ordinary skill in the art are familiar with numerous cloning vectors, and the selection of an appropriate cloning vector is a matter of choice. The construction of vectors containing desired nucleotide sequences linked by appropriate DNA sequences is accomplished by discussed above. These vectors may be constructed to contain additional DNA sequences, such as bacterial origins of replication to make shuttle vectors in order to shuttle between prokaryotic hosts and mammalian hosts.

Procedures for construction and expression of mutant proteins of defined sequence are well known in the art. A DNA sequence encoding a mutant form of VWF or a fragment thereof can be synthesized chemically or prepared from the wild-type sequence by one of several approaches, including primer extension, linker insertion and PCR (see, e.g., Sambrook, et al., (1989) Nature November 16; 342(6247):224-5; Maniatis, Fritsch & Sambrook, “Molecular Cloning: A Laboratory Manual” (1982): “DNA Cloning: A Practical Approach,” Volumes I and II (D. N. Glover, ed., 1985); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Nucleic Acid Hybridization” (B. D. Hames & S. J. Higgins, eds., 1985); “Transcription and Translation” (B. D. Hames & S. J. Higgins, eds., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1986); “Immobilized Cells and Enzymes” (IRL Press, 1986): B. Perbal, “A Practical Guide to Molecular Cloning” (1984), and Sambrook, et al., “Molecular Cloning: a Laboratory Manual” (1989)). Mutants can be prepared by these techniques having additions, deletions, and substitutions in the wild-type sequence (for example, the mouse VWF-A1 1326R>H mutant of SEQ ID NO: 5). To confirm that the mutant contains the desired changes, one skilled in the art can confirm the changes of interest via sequence-by-sequence analysis and/or by methods available to one skilled in the art.

In one embodiment, modification of the A1 domain can contain an amino acid residue substitution at a position involved with binding to GPIb alpha (such as, but not limited to, positions 1263, 1269, 1274, 1287, 1302, 1308, 1313, 1314, 1326, 1329, 1330, 1333, 1344, 1347, 1350, 1370, 1379, 1381, 1385 1391, 1394, 1397, 1421, 1439, 1442, 1449, 1466, 1469, 1472, 1473, 1475, 1479). In further embodiments, the modification of the A1 domain can be a partial or full replacement of an animal (such as a mouse) A1 domain of VWF with the A1 domain of human VWF. In other embodiments of the invention, the modification of the A1 domain can be an amino acid substitution at residue 1326 (for example HIS for ARG) such as depicted in SEQ ID NO: 5.

SEQ ID NO: 5 is the sequence for the mouse VWF-A1 1326 R>H mutant, wherein the modified amino acid sequence corresponds to the A1 domain of mouse VWF (residues 1260-1480) having an amino acid substitution at residue 1326 of HIS for ARG (in Bold):

EDTPEPPLHNFYCSKLLDLVFLLDGSSMLSEAEFEVLKAFVVGMMERLHI SQKRIRVAVVEYHDGS H AYLELKARKRPSELRRITSQIKYTGSQVASTSE VLKYTLFQIFGKIDRPEASHITLLLTASQEPPRMARNLVRYVQGLKKKKV IVIPVGIGPHASLKQIRLIEKQAPENKAFLLSGVDELEQRRDEIVSYLCD LAPEAPAPTQPPQVAHVTVSP

An expression vector containing a nucleotide sequence encoding a protein of interest, such as a mutant VWF-A1 molecule described above, is transfected into a host cell, either eukaryotic (for example, yeast, mammalian, or insect cells) or prokaryotic, by conventional techniques well established in the art. Transfection techniques carried out depend on the host cell used. For example, mammalian cell transfection can be accomplished using lipofection, protoplast fusion, DEAE-dextran mediated transfection, CaPO₄ co-precipitation, electroporation, direct microinjection, as well as other methods known in the art which can comprise: scraping, direct uptake, osmotic or sucrose shock, lysozyme fusion or erythrocyte fusion, indirect microinjection such as via erythrocyte-mediated techniques, and/or by subjecting host cells to electric currents. Some of the techniques mentioned above are also applicable to unicellular organisms, such as bacteria or yeast. As other techniques for introducing genetic information into host cells will be developed, the above-mentioned list of transfection methods is not considered to be exhaustive. The transfected cells are then cultured by conventional techniques to produce a mutant VWF-A1 molecule harboring at least one of the mutations previously described.

One skilled in the art understands that expression of desired protein products in prokaryotes is most often carried out in E. coli with vectors that contain constitutive or inducible promoters. Some non-limiting examples of bacterial cells for transformation include the bacterial cell line E. coli strains DH5α or MC1061/p3 (Invitrogen Corp., San Diego, Calif.), which can be transformed using standard procedures practiced in the art, and colonies can then be screened for the appropriate plasmid expression. Some E. coli expression vectors (also known in the art as fusion-vectors) are designed to add a number of amino acid residues, usually to the N-terminus of the expressed recombinant protein. Such fusion vectors can serve three functions: 1) to increase the solubility of the desired recombinant protein; 2) to increase expression of the recombinant protein of interest; and 3) to aid in recombinant protein purification by acting as a ligand in affinity purification. In some instances, vectors, which direct the expression of high levels of fusion protein products that are readily purified, may also be used. Some non-limiting examples of fusion expression vectors include pGEX, which fuse glutathione S-tranferase to desired protein; pcDNA 3.1V5-His A B & C (Invitrogen Corp, Carlsbad, Calif.) which fuse 6×-His to the recombinant proteins of interest; pMAL (New England Biolabs, MA) which fuse maltose E binding protein to the target recombinant protein; the E. coli expression vector pUR278 (Ruther et al., (1983) EMBO 12:1791), wherein the coding sequence may be ligated individually into the vector in frame with the lac Z coding region in order to generate a fusion protein; and pIN vectors (Inouye et al., (1985) Nucleic Acids Res. 13:3101-3109; Van Heeke et al., (1989) J. Biol. Chem. 24:5503-5509. Fusion proteins generated by the likes of the above-mentioned vectors are generally soluble and can be purified easily from lysed cells via adsorption and binding to matrix glutathione agarose beads subsequently followed by elution in the presence of free glutathione. For example, the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target can be released from the GST moiety.

Other suitable cell lines, in addition to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing coding sequences for a mutant VWF-A1 molecule described above, may alternatively be used to produce the molecule of interest. Non-limiting examples include plant cell systems infected with recombinant virus expression vectors (for example, tobacco mosaic virus, TMV; cauliflower mosaic virus, CaMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing coding sequences for a mutant VWF-A1 molecule described above; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing coding sequences for a mutant VWF-A1 molecule described above; yeast (for example, Saccharomyces sp., Pichia sp.) transformed with recombinant yeast expression vectors containing coding sequences for a mutant VWF-A1 molecule described above; or mammalian cell lines harboring a vector that contains coding sequences for a mutant VWF-A1 molecule described above.

Mammalian cells (such as BHK cells, VERO cells, CHO cells and the like) can also contain an expression vector (for example, one that harbors a nucleotide sequence encoding a mutant VWF-A1 molecule described above) for expression of a desired product. Expression vectors containing such a nucleic acid sequence linked to at least one regulatory sequence in a manner that allows expression of the nucleotide sequence in a host cell can be introduced via methods known in the art, as described above. To those skilled in the art, regulatory sequences are well known and can be selected to direct the expression of a protein of interest in an appropriate host cell as described in Goeddel, Gene Expression Technology (1990) Methods in Enzymology 185, Academic Press, San Diego, Calif.). Regulatory sequences can comprise the following: enhancers, promoters, polyadenylation signals, and other expression control elements. Practitioners in the art understand that designing an expression vector can depend on factors, such as the choice of host cell to be transfected and/or the type and/or amount of desired protein to be expressed.

Animal or mammalian host cells capable of harboring, expressing, and secreting large quantities of a mutant VWF-A1 molecule (described above) of interest into the culture medium for subsequent isolation and/or purification include, but are not limited to, Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCC CCL-61), DG44 (Chasin et al., (1986) Som. Cell Molec. Genet, 12:555-556; Kolkekar et al., (1997) Biochemistry, 36:10901-10909; and WO 01/92337 A2), dihydrofolate reductase negative CHO cells (CHO/dhfr-, Urlaub et al., (1980) Proc. Natl. Acad. Sci. U.S.A., 77:4216), and dp12.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651); human embryonic kidney cells (e.g., 293 cells, or 293 cells subcloned for growth in suspension culture, Graham et al., (1977) J. Gen. Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL-10); monkey kidney cells (CV1, ATCC CCL-70); African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse sertoli cells (TM4; Mather (1980) Biol. Reprod., 23:243-251); human cervical carcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TRI cells (Mather (1982) Annals NY Acad. Sci., 383:44-68); MCR 5 cells; FS4 cells. A cell line transformed to produce a mutant VWF-A1 molecule described above can also be an immortalized mammalian cell line of lymphoid origin, which include but are not limited to, a myeloma, hybridoma, trioma or quadroma cell line. The cell line can also comprise a normal lymphoid cell, such as a B cell, which has been immortalized by transformation with a virus, such as the Epstein Barr virus (such as a myeloma cell line or a derivative thereof).

A host cell strain, which modulates the expression of the inserted sequences, or modifies and processes the nucleic acid in a specific fashion desired also may be chosen. Such modifications (for example, glycosylation and other post-translational modifications) and processing (for example, cleavage) of protein products may be important for the function of the protein. Different host cell strains have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. As such, appropriate host systems or cell lines can be chosen to ensure the correct modification and processing of the foreign protein expressed, such as a mutant VWF-A1 molecule described above. Thus, eukaryotic host cells possessing the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Non-limiting examples of mammalian host cells include 3T3, W138, BT483, Hs578T, CHO, VERY, BHK, Hela, COS, BT2O, T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, MDCK, 293, HTB2, and HsS78Bst cells.

For protein recovery, isolation and/or purification, the cell culture medium or cell lysate is centrifuged to remove particulate cells and cell debris. The desired polypeptide molecule (for example, a mutant VWF-A1 protein) is isolated or purified away from contaminating soluble proteins and polypeptides by suitable purification techniques. Non-limiting purification methods for proteins include: separation or fractionation on immunoaffinity or ion-exchange columns; ethanol precipitation; reverse phase HPLC; chromatography on a resin, such as silica, or cation exchange resin, e.g., DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, e.g., Sephadex G-75, Sepharose; protein A sepharose chromatography for removal of immunoglobulin contaminants; and the like. Other additives, such as protease inhibitors (e.g., PMSF or proteinase K) can be used to inhibit proteolytic degradation during purification. Purification procedures that can select for carbohydrates can also be used, e.g., ion-exchange soft gel chromatography, or HPLC using cation- or anion-exchange resins, in which the more acidic fraction(s) is/are collected.

In one embodiment, the protein isolated is a mutant human von Willebrand Factor A1 protein comprising one or more of the following mutations: 1263S>P, 1269D>N, 1274R>K, 1287R>M, 1302D>G, 1308R>H, 1313W>R, 1314V>I, 1326H>R, 1329I>L, 1330G>E, 1333D>A, 1344A>T, 1347V>I, 1350A>T, 1370S>G, 1379R>H, 1381A>T, 1385M>T 1391Q>P, 1394S>A, 1397F>L, 1421N>S, 1439V>L, 1442S>G, 1449Q>R, 1466P>A, 1469L>Q, 1472H>Q, 1473M>V, 1475Q>H, or 1479G>S. In another embodiment, the protein isolated is a mutant human von Willebrand Factor A1 protein comprising a 1263S>P, 1269D>N, 1274R>K, 1287R>M, 1302D>G, 1308R>H, 1313W>R, 1314V>I, 1326H>R, 1329I>L, 1330G>E, 1333D>A, 1344A>T, 1347V>I, 1350A>T, 1370S>G, 1379R>H, 1381A>T, 1385M>T, 1391Q>P, 1394S>A, 1397F>L, 1421N>S, 1439V>L, 1442S>G, 1449Q>R, 1466P>A, 1469L>Q, 1472H>Q, 1473M>V, 1475Q>H, or a 1479G>S mutation. In a particular embodiment, the protein isolated is a mutant human von Willebrand Factor A1 protein comprising a 1326H>R mutation.

The invention also provides a method for producing mutant von Willebrand Factor A1 protein that specifically binds human platelets. For example, an animal expressing a mutant von Willebrand Factor A1 (VWF-A1) protein can be provided, wherein the mutation causes the platelet binding specificity of the animal VWF-A1 protein to change to be specific for human platelets. VWF plasma protein containing the mutant A1 domain from an animal (such as from a mouse) can then be subsequently harvested. In one embodiment, the animal von Willebrand Factor A1 protein contains at least one mutation at amino acid position 1263, 1269, 1274, 1287, 1302, 1308, 1313, 1314, 1326, 1329, 1330, 1333, 1344, 1347, 1350, 1370, 1379, 1381, 1385, 1391, 1394, 1397, 1421, 1439, 1442, 1449, 1466, 1469, 1472, 1473, 1475, or 1479. In another embodiment, the mutations occur in a murine VWF-A1 protein. In particular embodiments, the mutant murine von Willebrand Factor A1 protein comprises at least one mutation comprising 1263P>S, 1269N>D, 1274K>R, 1287M>R, 1302G>D, 1308H>R, 1313R>W, 1314I>V, 1326R>H, 1329L>I, 1330E>G, 1333A>D, 1344T>A, 1347I>V, 1350T>A, 1370G>S, 1379H>R, 1381T>A, 1385T>M 1391P>Q, 1394A>S, 1397L>F, 1421S>N, 1439L>V, 1442G>S, 1449R>Q, 1466A>P, 1469Q>L, 1472Q>H, 1473V>M, 1475H>Q, 1479S>G, or any combination thereof.

Pre-Screening Evaluation of Anti-Thrombotics and Associated Diseases

Diversity libraries, such as random or combinatorial peptide or non-peptide libraries can be screened for small molecules and compounds that specifically bind to a VWF-A1 protein. Many libraries are known in the art that can be used such as, e.g., chemically synthesized libraries, recombinant (e.g., phage display) libraries, and in vitro translation-based libraries.

Any screening technique known in the art can be used to screen for agonist (i.e., compounds that promote platelet adhesion) or antagonist molecules (such as anti-thrombotics) directed at a target of interest (e.g. VWF-A1). The present invention contemplates screens for small molecule ligands or ligand analogs and mimics, as well as screens for natural ligands that bind to and modulate VWF-A1 binding to GPIb-alpha, such as via examining the degree of thrombus formation, platelet adhesion, coagulation, blood flow, vessel occlusion, or bleeding times. For example, natural products libraries can be screened using assays of the invention for molecules that modulate the activity of a molecule of interest, such as a VWF-A1 binding to GPIb-alpha.

Knowledge of the primary sequence of a molecule of interest, such as a VWF-A1, can provide an initial clue as to proteins that can modulate VWF-A1 binding to GPIb-alpha. Identification and screening of modulators is further facilitated by determining structural features of the protein, e.g., using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry, and other techniques for structure determination. These techniques provide for the rational design or identification of such modulators.

Test compounds, such as test modulators of VWF-A1 binding to GPIb-alpha, are screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, Wash.) or MycoSearch (N.C.), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., (1996) Tib Tech 14:60).

Methods for preparing libraries of molecules are well known in the art and many libraries are commercially available. Libraries of interest in the invention include peptide libraries, randomized oligonucleotide libraries, synthetic organic combinatorial libraries, and the like. Degenerate peptide libraries can be readily prepared in solution, in immobilized form as bacterial flagella peptide display libraries or as phage display libraries. Peptide ligands can be selected from combinatorial libraries of peptides containing at least one amino acid. Libraries can be synthesized of peptoids and non-peptide synthetic moieties. Such libraries can further be synthesized which contain non-peptide synthetic moieties, which are less subject to enzymatic degradation compared to their naturally-occurring counterparts. Libraries are also meant to include for example but are not limited to peptide-on-plasmid libraries, polysome libraries, aptamer libraries, synthetic peptide libraries, synthetic small molecule libraries and chemical libraries. The libraries can also comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the above-identified functional groups. Screening compound libraries listed above [also see EXAMPLE 6 and U.S. Patent Application Publication No. 2005/0009163, which is hereby incorporated by reference], in combination with dynamic force microscopy, a coagulation factor assay, a platelet adhesion assay, thrombus imaging, a bleeding time assay, aggregometry, review of real-time video of blood flow, a Doppler ultrasound vessel occlusion assay, or a combination of these assays (for example, those assays described in EXAMPLES 1-4) can be used to identify modulators of VWF-A1 binding to GPIb-alpha, wherein the compound abbreviates or increases off-rate (k_(off)) binding kinetics between VWF-A1 and GPIb-alpha by at least two-fold (Lew et al., (2000) Curr. Med. Chem. 7(6):663-72; Werner et al., (2006) Brief Funct. Genomic Proteomic 5(1):32-6).

Small molecule combinatorial libraries may also be generated. A combinatorial library of small organic compounds is a collection of closely related analogs that differ from each other in one or more points of diversity and are synthesized by organic techniques using multi-step processes. Combinatorial libraries include a vast number of small organic compounds. One type of combinatorial library is prepared by means of parallel synthesis methods to produce a compound array. A compound array can be a collection of compounds identifiable by their spatial addresses in Cartesian coordinates and arranged such that each compound has a common molecular core and one or more variable structural diversity elements. The compounds in such a compound array are produced in parallel in separate reaction vessels, with each compound identified and tracked by its spatial address. Examples of parallel synthesis mixtures and parallel synthesis methods are provided in U.S. Ser. No. 08/177,497, filed Jan. 5, 1994 and its corresponding PCT published patent application WO95/18972, published Jul. 13, 1995 and U.S. Pat. No. 5,712,171 granted Jan. 27, 1998 and its corresponding PCT published patent application WO96/22529, which are hereby incorporated by reference.

Examples of chemically synthesized libraries are described in Fodor et al., (1991) Science 251:767-773; Houghten et al., (1991) Nature 354:84-86; Lam et al., (1991) Nature 354:82-84; Medynski, (1994) BioTechnology 12:709-710; Gallop et al., (1994) J. Medicinal Chemistry 37(9):1233-1251; Ohlmeyer et al., (1993) Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., (1994) Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al., (1992) Biotechniques 13:412; Jayawickreme et al., (1994) Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et al., (1993) Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT Publication No. WO 93/20242, dated Oct. 14, 1993; and Brenner et al., (1992) Proc. Natl. Acad. Sci. USA 89:5381-5383.

Screening methods of the invention allowed for the identification of potential compounds that modulate VWF-A1 binding to GPIb-alpha. In some embodiments of the invention, the compound comprises one or more compounds having a structure depicted in Table 8 below.

Examples of phage display libraries are described in Scott et al., (1990) Science 249:386-390; Devlin et al., (1990) Science, 249:404-406; Christian, et al., (1992) J. Mol. Biol. 227:711-718; Lenstra, (1992) J. Immunol. Meth. 152:149-157; Kay et al., (1993) Gene 128:59-65; and PCT Publication No. WO 94/18318.

In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058; and Mattheakis et al., (1994) Proc. Natl. Acad. Sci. USA 91:9022-9026.

In one non-limiting example, non-peptide libraries, such as a benzodiazepine library (see e.g., Bunin et al., (1994) Proc. Natl. Acad. Sci. USA 91:4708-4712), can be screened. Peptoid libraries, such as that described by Simon et al., (1992) Proc. Natl. Acad. Sci. USA 89:9367-9371, can also be used. Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994), Proc. Natl. Acad. Sci. USA 91:11138-11142.

Screening the libraries can be accomplished by any variety of commonly known methods. See, for example, the following references, which disclose screening of peptide libraries: Parmley and Smith, (1989) Adv. Exp. Med. Biol. 251:215-218; Scott and Smith, (1990) Science 249:386-390; Fowlkes et al., (1992) BioTechniques 13:422-427; Oldenburg et al., (1992) Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., (1994) Cell 76:933-945; Staudt et al., (1988) Science 241:577-580; Bock et al., (1992) Nature 355:564-566; Tuerk et al., (1992) Proc. Natl. Acad. Sci. USA 89:6988-6992; Ellington et al., (1992) Nature 355:850-852; U.S. Pat. Nos. 5,096,815; 5,223,409; and 5,198,346, all to Ladner et al.; Rebar et al., (1993) Science 263:671-673; and PCT Pub. WO 94/18318.

The invention provides a method for identifying a compound that modulates VWF-A1 binding to GPIb-alpha. In one embodiment, the method can comprise providing an electronic library of test compounds stored on a computer (such as those libraries described above); providing atomic coordinates for at least 10 amino acid residues of the A1 domain of the VWF protein listed in Table 8, where the coordinates having a root mean square deviation therefrom, with respect to at least 50% of the Cα atoms, of not greater than about 2.5 Å, in a computer readable format; converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the VWF-A1 domain; performing a data processing method, wherein electronic test compounds from the library are superimposed upon the three dimensional model of the A1 domain of VWF; and determining which test compound fits within the binding pocket of the three dimensional model of the VWF-A1 protein. Thus, compounds can be identified that would modulate the binding of VWF-A1 to GPIb-alpha.

In another embodiment, the method can comprise providing an electronic library of test compounds stored on a computer; and providing atomic coordinates listed in Table 8 in a computer readable format for at least 10, 15, 20, 25, 30, 35, or 40 amino acid residues of the A1 domain of the VWF protein, wherein the residues can comprise 2 or more of the following residues: Pro1391, Arg1392, Arg1395, Val1398, Arg1399, Gln1402, Lys1406, Lys1423, Gln1424, Leu1427, Lys1430, or Glu1431; converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the A1 domain of the VWF protein; performing a data processing method, wherein electronic test compounds from the library are superimposed upon the three dimensional model of the A1 domain of the VWF protein; and determining which test compound fits within the binding pocket of the three dimensional model of the VWF-A1 protein. Thus, compounds can be identified that would modulate the binding of VWF-A1 to GPIb-alpha.

In a further embodiment, the method can comprise providing an electronic library of test compounds stored on a computer (such as those libraries described above); providing atomic coordinates for at least 10 amino acid residues of the Botrocetin-VWF-A1 complex listed in accession entry 1IJK (http://www.rcsb.org/pdb/explore.do?structureId=1IJK), where coordinates having a root mean square deviation therefrom, with respect to at least 50% of the Cα atoms, not more than about 3 Å, in a computer readable format; converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the Botrocetin-VWF-A1 complex; performing a data processing method, wherein electronic test compounds from the library are superimposed upon Botrocetin within the three dimensional model of the Botrocetin-VWF-A1 complex; and determining which test compound fits within the binding pocket of the three dimensional model of the VWF-A1 protein and best overlays the three-dimensional model generated above. Thus, compounds can be identified that would modulate the binding of VWF-A1 to GPIb-alpha.

In other embodiments, the method can comprise providing an electronic library of test compounds stored on a computer; providing atomic coordinates listed in accession entry 1IJK (http://www.rcsb.org/pdb/explore.do?structureId=1IJK) in a computer readable format for at least 10, 15, 20, 25, 30, 35, or 40 amino acid residues of the Botrocetin-murine VWF-A1 complex, wherein the residues comprise 2 or more of the following residues: Pro 1391, Arg1392, Arg1395, Val1398, Arg1399, Gln1402, Lys1406, Lys1423, Gln1424, Leu1427, Lys1430, or Glu1431; converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the Botrocetin-VWF-A1 complex; performing a data processing method, wherein electronic test compounds from the library are superimposed upon Botrocetin within the three dimensional model of the Botrocetin-VWF-A1 complex; and determining which test compound fits within the binding pocket of the three dimensional model of the VWF-A1 protein and best overlays the three-dimensional model generated above. Thus, compounds can be identified that would modulate the binding of VWF-A1 to GPIb-alpha.

The invention also provides for a compound identified by the method described above. In one embodiment, the compound inhibits thrombosis formation or promotes platelet adhesion.

The present invention provides methods for evaluating potential anti-thrombotic reagents in pre-clinical testing using a non-human transgenic animal (for example, non-human animals include, but are not limited to, vertebrates such as ovines, bovines, rodents, non-human primates, porcines, caprines, equines, ruminants, lagomorphs, canines, felines, aves, and the like). There are three main classes of antithrombotic drugs that can be screened using the transgenic mouse model of the invention: Anticoagulant drugs (such as Heparins; Vitamin K antagonists, which are currently the only anticoagulants that can be administered orally; and direct thrombin inhibitors), Antiplatelet drugs (such as cyclooxygenase inhibitors like aspirin; phosphodiesterase inhibitors like ticlopidine (Ticlid); adenosine diphosphate receptor inhibitors like clopidogrel (Plavix); tirofiban (Aggrastat); adenosine reuptake inhibitors, and inhibitors of integrins on platelets (for example, alpha IIb Beta3) like eptifibatide (Integrilin)), and Thrombolytic or fibrinolytic drugs (such as t-PA (alteplase Activase); reteplase (Retavase); urokinase (Abbokinase); streptokinase (Kabikinase, Streptase); tenectaplase; lanoteplase; and anistreplase (Eminase)).

The invention provides an in vivo model to test the efficacy of potential anti-thrombotic drugs against human platelets prior to FDA approval. To date, in vitro models of thrombosis do not accurately recapitulate the hemodynamic conditions, cell-cell interactions, or cell-protein interactions that occur at sites of vascular injury in a living animal. Thus, anti-thrombotics can be identified and their potential therapeutic effects can be assessed for treatment of abnormal thrombotic events associated with atherothrombotic arterial diseases and venous thrombotic diseases (such as abnormal bleeding and/or abnormal clotting).

Atherothrombotic arterial diseases can include, but is not limited to, coronary artery disease, (for example, acute myocardial infarction, acute coronary syndromes (such as unstable angina pectoris) and stable angina pectoris); mesenteric ischemia, “abdominal angina,” and mesenteric infarction; cerebral vascular disease, including acute stroke and transient ischemic attack; mesenteric arterial disease; as well as peripheral arterial disease, including acute peripheral arterial occlusion and intermittent claudication. Anti-thrombotic compounds identified by the pre-clinical testing method of the present invention can also be useful for the treatment of coronary artery disease (which includes, but is not limited to anti-thrombotic therapy during coronary angioplasty, anti-thrombotic therapy during cardiopulmonary bypass, and limiting of platelet activation during ischemia reperfusion) as well as venous thrombotic diseases (which include, but are not limited to deep venous thrombosis and pulmonary thromboembolism). Anti-thrombotic compounds identified by the pre-clinical testing method of the present invention can also be useful in anti-thrombotic therapy for pulmonary hypertension.

The invention provides a method for testing a compound that modulates VWF-A1 binding to GPIb-alpha. The method can entail obtaining or synthesizing a compound identified in the screens previously described above; contacting VWF-A1 with the compound under a condition suitable for GPIb-alpha-VWF-A1 binding; and determining whether the compound can modulate GPIb-alpha-VWF-A1 binding using a diagnostic assay. In one embodiment, contacting can comprises perfusing platelets into a flow chamber at a shear flow rate of at least 100 s⁻¹, wherein mutant murine VWF-A1 protein is immobilized on a bottom surface of the chamber. In another embodiment, contacting can comprise perfusing platelets into a transgenic non-human, for example the transgenic mouse described in EXAMPLE 3. In some embodiments, of the invention, contacting first occurs in vitro by way of the flow chamber described above, subsequently followed by in vivo testing of the compound's efficiency to modulate GPIb-alpha binding to VWF-A1 after the compound was determined to have a purported effect in modulating such binding in vitro. Thus, the invention provides a great advantage of being able to test directly compounds that target human platelets in an in vivo system. The transgenic mouse (which, for example, can harbor the 1326R>H mutation in the A1 domain of VWF of SEQ ID NO: 5) displays a bleeding phenotype, thus serves as a model for screening potential anti-thrombotic compounds useful for humans when the mouse is perfused with human platelets. Since the 1326R>H mutation in VWF-A1 in the mouse model (SEQ ID NO: 5) has been shown to support human platelet binding and it corresponds to the His amino acid at the same position in human VWF-A1 (as well as in canines, chimpanzees, rat, porcine, felines, equines, bovine, and the like (Jenkins et al., (1998) Blood 91(6): 2032-44)), the test compounds screened using this mouse model (while subject to perfusion with platelets from human, dog, cat, or other relevant organism) will be applicable to multiple species.

After perfusion with human platelets, a test compound (such as a purported anti-thrombotic that would minimize blood clotting or a compound that could promote platelet adhesion) can be administered to the animal subsequent to vessel injury in order to determine whether blood clotting is minimized or if it is enhanced. In one embodiment, the platelets infused are human platelets while in other embodiments the platelets infused are not murine platelets. In some embodiments, the compound can slow the on-rate, and/or increase the off-rate (k_(off)) binding kinetics, and/or reduce bond strength of the interaction between VWF-A1 and GPIb-alpha by at least two-fold, thus resulting in a decreased lifetime of the bond(s). Such compounds could reduce thrombosis formation. In other embodiments, the compound can abbreviate off-rate (k_(off)) binding kinetics between VWF-A1 and GPIb-alpha by at least two-fold, thus resulting in a prolongation in the lifetime of the bond(s). Such compounds could promote platelet adhesion due to the compound stabilizing an interaction between VWF-A1 and GPIb-alpha. To assess binding efficiency between VWF-A1 and GPIb-alpha, binding kinetics can be determined by measuring translocation velocity, tethering frequency, and bond strength (Fukuda, K., et al., (2005) Nat. Struct. Mol. Biol. 12:152-159; Doggett, et al., (2003) Blood 102(10): 152-60; Doggett, T. A. et al. (2002) Biophys. J. 83, 194-205; Schmidtke and Diamond (2000) J Cell Bio 149(3): 719-29; Mody et al., (2005) Biophys. J. 88: 1432-43, all of which are incorporated by reference in their entirety).

The compound identified and tested using the methods described above can be an anti-platelet drug. In one embodiment, the anti-platelet drug can be a cyclooxygenase inhibitor, a phosphodiesterase inhibitor, an adenosine diphosphate receptor inhibitor, a PI3K inhibitor, an adenosine reuptake inhibitors, thrombin receptor inhibitor or inhibitor of any intracellular signaling pathway in platelets, an alphaIIb beta3 inhibitor, an alpha2 beta1 inhibitor, a glycoprotein V inhibitor, a glycoprotein VI inhibitor, a PECAM-1 inhibitor or any adhesion molecule and/or activation pathway critical for human platelet function

The diagnostic assay used in this method for testing a compound that modulates VWF-A1 binding to GPIb-alpha can assess whether an abnormal thrombotic event occurred in the subject. An abnormal thrombotic event can comprise abnormal bleeding, abnormal clotting, death, or a combination thereof. The assay can comprise dynamic force microscopy, a coagulation factor assay, a platelet adhesion assay, thrombus imaging, a bleeding time assay, aggregometry, review of real-time video of blood flow, a Doppler ultrasound vessel occlusion assay, or a combination thereof.

After perfusion with a purported anti-thrombotic or a compound that could promote platelet adhesion, a labeled agent can subsequently be perfused either into the flow chamber or to the animal. Such an agent would enable the visualization of either the presence or absence of a thrombus. In one embodiment, the labeled agent can comprise one or more of a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, or a small molecule ligand. In some embodiments, the agent can target a platelet receptor, a VWF protein, or a portion thereof.

Methods for Assessing Thrombotic Events In Vivo

The invention provides methods for detecting an internal vascular injury site (occult bleeding) in a subject. This could be useful in ER settings or on the battlefield in order to quickly identify sites of internal bleeding. For instance, the method can entail: administering to a subject a targeted molecular imaging agent, wherein the molecule circulates for an effective period of time in order to bind to the injury site within the subject; tracking a deposition of the labeled thrombosis-indicating-molecule in the subject; and identifying the site of a thrombus formation in the subject by imaging the labeled targeted molecular imaging agent. Thus, the deposition of the targeted molecular imaging agent at the internal vascular injury site can be indicative of internal bleeding within a subject. For example, a targeted molecular imaging agent can recognize constituents of thrombi that comprise a lipid, a protein, a cellular molecule, or a combination thereof. In one embodiment, the targeted molecular imaging agent is administered by subcutaneous, intra-muscular, intra-peritoneal, or intravenous injection; infusion; by oral, nasal, or topical delivery; or a combination of the listed routes of administration. In other embodiments, the targeted molecular imaging agent has a T_(1/2) of at least 30 min. In some embodiments, the targeted molecular imaging agent can comprise an antibody, peptide, or Fab fragment directed to a platelet receptor, a VWF protein, or a portion thereof. In particular embodiments, the targeted molecular imaging agent can comprise a VWF-A1 or GPIb-alpha receptor trap. For example, a receptor trap is a decoy receptor that can comprise fusions between two distinct receptor components and the Fc region of an antibody molecule, which can result in the generation of a molecule with an increased affinity over single component reagents. This technology is available from Regeneron (Tarrytown, N.Y.) and is described in Wachsberger et al., (2007) Int J Radiat Oncol Biol Phys. 67(5):1526-37; Holash et al., (2002) Proc Natl Acad Sci U S A. 2002 99(17):11393-8; Davis et al., (1996) Cell. 87(7):1161-9; U.S. Pat. No. 7,087,411; and in United States Publication Applications 2004/0014667, 2005/0175610, 2005/0260203, 2006/0030529, 2006/0058234, which are all hereby incorporated by reference in their entirety.

To aid in the visualization of a site of thrombus formation, the targeted molecular imaging agent can further comprise a label. In one embodiment, the labeled thrombosis-indicating-molecule comprises a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, a peptide, a small molecule ligand, or a combination thereof. A fluorophore, for example green fluorescent protein, (such as GFP, RFP, YFP and the like; see Johnson and Johnson, (2007) ACS Chem. Biol. 2(1):31-8) can be used as a biomarker. A quantum dot is a semiconductor nanocrystal, that can be as small as 2 to 10 nm or can 15-20 nm (for example, Q-dot nanocrystals; also see Kaji et al., (2007) Anal Sci. 23(1):21-4). Quantum dot fluorescence can be induced by exposure to ultraviolet light. Both a fluorescent protein and a quantum dot can be obtained commercially (for example, Molecular Probes—Invitrogen, Carlsbad, Calif. or Evident Technologies, Troy N.Y.). A fluorophore can also be generated in the laboratory according to molecular biology methods practiced in the art. A radiolabel is a radioactive isotope that can be used as a tracer. Non-limiting examples of radiolabels include: Technetium-99m, Iodine-123 and 131, Thallium-201, Gallium-67, Fluorine-18, -19, Indium-111, Xenon-I 33, and Krypton-81m. Radiolabels can be obtained commercially, for example, from SRI International (Menlo Park, Calif.). In one embodiment, the nanoparticle can comprise a perfluorocarbon (PFC). Non-limiting examples of perfluorocarbons include perfluorobutane, perfluorohexane, perfluorooctane, perfluorodecalin, perfluoromethyldecalin, and perfluoroperhydrophenanthrene. These can be synthesized according to the method described in EXAMPLE 5 or according to Partlow et al. (FASEB J (2007) February 6 on-line publication, fj.06-6505com). The perfluorocarbon molecules can also be obtained commercially (F2 Chemicals Ltd.; Lancashire, UK). In another embodiment, the PFC nanoparticle can be coupled to a platelet receptor antibody (such as platelet receptor alpha-IIb beta₃). In some embodiments, imaging can comprise a PET scan, a CT scan, an MRI, an IR scan, an ultrasound, nuclear imaging, or a combination thereof.

Since the usefulness of the method pertains to the swiftness in identifying sites of internal bleeding (for example in an ER setting or on the battlefield), the subject can be further administered a compound aid in the cessation of such bleeding. In one embodiment, the subject is further administered a thrombotic compound (for example, a compound identified in the screens described above or a compound comprising a structure depicted in Table 8). In some embodiments, the compound can abbreviate off-rate (k_(off)) binding kinetics, and/or slow the on-rate, and/or reduce the bond strength between VWF-A1 and GPIb-alpha by at least two-fold.

The invention also provides a method to test contrast agents for imaging of human platelets at sites of thrombosis. For instance, one could test the ability of nanoparticle contrast agents targeted to human platelets to identify areas of thrombosis or occult bleeding. In some embodiments, the prevention or reduction of thrombus formation at site of injury upon administration of a compound can be visually examined via tracking the localization of labeled platelets (such as with high resolution in vivo microscopy or MRI). In further embodiments, the platelets can be labeled with a nanoparticle, fluorophore, quantum dot, microcrystal, radiolabel, dye, or gold biolabel. The prevention or reduction of thrombus formation also can be readily determined by methods known to one skilled in the art, which include but are not limited to aggregometry, review of real-time video of blood flow in the animal, and determination of vessel occlusion, as well as by the examples provided below.

Additionally, the transgenic mouse's bleeding phenotype can be exploited to screen potential prothrombotic compounds, in addition to anti-thrombotics discussed above. A test compound (such as an alleged thrombotic that would induce and/or stimulate blood clotting) can be administered to the animal perfused with human platelets subsequent to vessel injury in order to determine whether blood clotting occurs. In some embodiments, the induction or stimulation of thrombus formation at site of injury upon administration of a compound can be visually examined via tracking the localization of labeled platelets (such as with high resolution in vivo microscopy or MRI). In further embodiments, the platelets can be labeled with a nanoparticle, fluorophore, quantum dot, microcrystal, radiolabel, dye, or gold biolabel. The prevention or reduction of thrombus formation also can be readily determined by methods known to one skilled in the art, which include but are not limited to aggregometry, ex-vivo flow chamber studies, review of real-time video of blood flow in the animal, and determination of vessel occlusion.

One of ordinary skill in the art can assess that the VWF-A1 mutants of this invention have the required properties of competitive binding to the GPIb platelet receptor in a manner that competes with the native VWF. Suitable assays are set forth in detail in the examples below, including ristocetin-induced platelet aggregation, platelet aggregation induced by ADP, thrombin, collagen, and platelet adhesion in a flow model.

In one embodiment of the invention, the non-human transgenic animal that expresses a modified A1 domain of the VWF sequence (for example, an amino acid residue substitution at a position involved with binding to GPIb alpha, such as, but not limited to, positions 1263, 1269, 1274, 1287, 1302, 1308, 1313, 1314, 1326, 1329, 1330, 1333, 1344, 1347, 1350, 1370, 1379, 1381, 1385 1391, 1394, 1397, 1421, 1439, 1442, 1449, 1466, 1469, 1472, 1473, 1475, 1479) can be used to validate new devices aimed at determining the effectiveness of antithrombotics in humans.

The non-human transgenic animal may also be used for determining the effectiveness of gene therapy (for example, assessing whether VWF-A1 protein targeting and protein expression was successful). Gene therapy refers to the insertion of genes into an individual's cells and tissues to treat a disease. For example, in a hereditary disease, a defective mutant allele is replaced with a functional one. The efficiency of VWF-A1 gene transfer by nonviral methodologies (i.e. lipofection) or viral methodologies (such as adenovirus infection described in U.S. Pat. No. 6,927,278 or United States Application publication No. 2005/0169899) can be assessed using the non-transgenic mouse model described above via examining whether replacement of a portion or the whole VWF gene in a subject (such as a mutant VWF mouse of the invention) affects clot formation in vivo. Results obtained from such a mouse model can then be correlated with the likely effect to be observed in human subjects. For gene therapy reviews, see Zuckerbraun et al., (2002) Arch Surg. 137(7):854-61; Melo et al., (2004) Arterioscler Thromb Vasc Biol. 24(10): 1761-74; and Dulak et al., (2006) Cell Biochem Biophys. 44(1):31-42, which are incorporated by reference in their entirety.

The invention provides a method for testing the efficiency of gene therapy in regulating thrombus formation in a subject. It also provides a method to test gene therapies directed at correcting genetic mutations associated with von Willebrand disease. The method can comprise the following steps: introducing a vector into the non-human transgenic animal of the invention described above, wherein the vector comprises a nucleic acid encoding a platelet receptor polypeptide, a platelet ligand polypeptide, or a VWF polypeptide, or a portion thereof; allowing sufficient time for expression of the polypeptide; perfusing platelets into the non-human transgenic animal that has one or more mutations in the VWF-A1 domain as previously described under a condition suitable for GPIb-alpha-VWF-A1 protein binding; and identifying an occurrence of a thrombotic event in the animal. For example, the vector introduced into the subject can be an adenovirus or DNA vector described in earlier sections utilizing methods discussed previously (see also Zuckerbraun et al., (2002) Arch Surg. 137(7):854-61; Melo et al., (2004) Arterioscler Thromb Vasc Biol. 24(10): 1761-74; and Dulak et al., (2006) Cell Biochem Biophys. 44(1):31-42). For example, the non-human animal that has one or more mutations in the VWF-A1 domain can be the murine model homozygous for the VWF-A1^(1326R>H) mutation.

In one embodiment of the invention, the platelets can be human platelets. In particular, the platelets are not murine platelets. In some embodiments, the thrombotic event comprises blood clotting, abnormal bleeding, abnormal clotting, death, or a combination thereof. Such an event can be identified using dynamic force microscopy, a coagulation factor assay, a platelet adhesion assay, thrombus imaging, a bleeding time assay, aggregometry, review of real-time video of blood flow, a Doppler ultrasound vessel occlusion assay, or a combination of the techniques previously described. In a further embodiment, perfusing platelets can be followed by a perfusion of a labeled agent. Non-limiting examples of a labeled agent comprises one or more of a nanoparticle, a fluorophore, a quantum dot, a micro crystal, a radiolabel, a dye, a gold biolabel, an antibody, or a small molecule ligand. In some embodiments, the agent targets a platelet receptor, a VWF protein, or a portion thereof.

The invention also provides a method to correlate results obtained with an in vitro assay designed to measure the effects of antithrombotics or biomarkers of platelet activation in patients. For example, a biomarker is an indicator of a particular disease state or a particular state of an organism, such as when the subject experiences vascular vessel wall injury. Upon injury to the vessel wall and subsequent damage to the endothelial lining, exposure of the subendothelial matrix to blood flow results in deposition of platelets at the site of injury via binding to the collagen with the surface collagen-specific glycoprotein Ia/IIa receptor. This adhesion is strengthened further by the large multimeric circulating protein VWF, which forms links between the platelet glycoprotein Ib/IX/V and collagen fibrils. The platelets are then activated and release the contents of their granules into the plasma, in turn activating other platelets. For example, Glycoprotein VI (GP6) is a 58-kD platelet membrane glycoprotein that plays a crucial role in the collagen-induced activation and aggregation of platelets. The shedding of GP6 can act as a marker representing that a person is at risk of myocardial infarction. In one embodiment, platelets obtained from a subject determined to have an elevated biomarker level (for example, GP6) can be infused into the non-human transgenic animal described above according to previously described methods, wherein the occurrence of a thrombotic event can be evaluated. In another embodiment, platelets obtained from a subject undergoing an anti-thrombotic treatment can be infused into the non-human transgenic animal described above according to previously described methods, wherein the occurrence of a thrombotic event can then be evaluated.

Method of Screening and Treating Subjects with Abnormalities of Platelet Function

The invention provides methods for treating subject with platelet function abnormalities, such as Von Willebrand disease (VWD), Bernard-Soulier syndrome, May-Hegglin anomaly, Chediak Higashi syndrome, and the like. In addition, the invention also provides methods for detecting abnormal platelet function or morphology in a subject.

VWD is a common hereditary coagulation abnormality that arises from a quantitative or qualitative deficiency of VWF). VWD affects humans, in addition to dogs and cats. There are three types of VWD: type 1, type 2, and type 3. Type 1 VWD is a quantitative defect, wherein decreased levels of VWF are detected but subjects may not have clearly impaired clotting, Type 2 VWD is a qualitative defect, wherein subjects have normal VWF levels but VWF multimers are structurally abnormal, or subgroups of large or small multimers are absent. Four subtypes exist: Type 2A, Type 2B, Type 2M, and Type 2N. Type 3 is rare and the most severe form of VWD (homozygous for the defective gene). (Braunwald et al., Harrison's Principle of Internal Medicine, 15 th ed., (Chapter 116) 2001, McGraw Hill, Columbus, Ohio).

Bernard-Soulier Syndrome is a rare disorder caused by a deficiency of the surface platelet receptor GPIb alpha. As a result, platelets fail to stick and clump together at the site of the injury. Functional abnormalities have also been observed in some hereditary platelet disorders wherein the platelets are of abnormal size or shape, such as in May-Hegglin Anomaly and Chediak Higashi syndrome. (Braunwald et al., Harrison's Principle of Internal Medicine, 15^(th) ed. (Chapter 116) 2001, McGraw Hill, Columbus, Ohio).

According to the invention, abnormal platelet function or morphology can be screened in a subject. The method can comprise the following steps: affixing a VWF-A1 molecule to a bottom surface of a flow chamber, or chip (such as a BIAcore chip), wherein the VWF-A1 molecule comprises at least one mutation at a position selected from the group consisting of 1263>S, 1269>D, 1274>R, 1287>R, 1302>D, 1308>R, 1313R>W, 1314>V, 1326>H, 1329>I, 1330>G, 1333>D, 1344>A, 1347>V, 1350>A, 1370>S, 1379>R, 1381>A, 1385>M 1391>Q, 1394>S, 1397>F, 1421>N, 1439>V, 1442>S, 1449>Q, 1466>P, 1469>L, 1472>H, 1473>M, 1475>Q, 1479>G, and any combination thereof, where the position corresponds to an amino acid position of human von Willebrand Factor A1 protein shown in SEQ ID NO: 6; perfusing a volume of whole blood or plasma over the surface-immobilized VWF-A1 molecule complexed to the murine mutant VWF-A1 protein in the flow chamber at a shear flow rate of at least 100 s⁻¹; perfusing a targeted molecular imaging agent into the flow chamber at a shear flow rate of at least 100 s⁻¹; determining whether platelets bind to the surface-immobilized-mutant-murine-VWF-A1 using a diagnostic assay; and comparing diagnostic assay results to a standard control, wherein the standard control sample was subjected to the steps described above. For example, the VWF molecule can be an antibody, a peptide, or a Fab fragment directed to a VWF polypeptide or a portion thereof. In one embodiment, the molecule can comprise a native or mutant VWF-A1 molecule, a purified native VWF or a mutant plasma VWF.

In one embodiment, whole blood or plasma sample can be perfused into the chamber or onto the chip, wherein the sample is obtained from the subject. For example, approximately 50 μl of whole blood can be perfused according to the method, or about 100 μl to about 150 μl of plasma can be perfused. As a standard control, the steps of the method described above can be performed using lyophilized non-self platelets, and can be subsequently compared to results obtained using the subjects' platelets. Here, the subject can be a human, a canine, a feline, a murine, a porcine, an equine, or a bovine.

In one embodiment, the targeted molecular imaging agent can comprise a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, a peptide, a small molecule ligand, or a combination thereof. In another embodiment, the targeted molecular imaging agent can bind to a platelet receptor, a platelet ligand, or any region of a VWF protein or a portion thereof. In a further embodiment, the targeted molecular imaging agent can comprise horseradish peroxidase (HRP) coupled to an antibody directed at VWF-A1. Following binding to VWF-A1, a reaction with diaminobenzidine (DAB) can be performed where DAB is reduced by HRP to produce a brown precipitate at the site of binding. This technique allows for enzymatic, calorimetric detection of binding that can be visualized by transmitted light microscopy. For example, if the antibody is directed at a platelet receptor, and calorimetric detection represents whether the antibody bound to the platelet-VWF-A1 complex, the absence of color would denote the lack of a complex formation, thus suggesting that platelets were unable to bind to VWF-A1. The lack of platelet binding could suggest functional defects in the subject's platelets. In one embodiment, platelets bound to VWF-A1 are less than about 500 cells/mm².

The normal platelet morphology is discoid with some spherical shaping. In one embodiment of the invention, the platelets obtained from the subject and that are subsequently screened are substantially spherical in shape. To further analyze platelet morphology, gross platelet histology can be assessed via light microscopy or electron microscopy. In another embodiment, platelets having an abnormal morphology are greater than about 2 μm in diameter. (Ross M H, Histology: A text and atlas 3^(rd) edition, Williams and Wilkins, 1995: Chapter 9). Various assays can be used to assess whether platelet function is normal, such as a platelet adhesion assay, fluorescence imaging, a chromogenic indication, microscopy morphology analysis, or those listed in Harrison's Principle of Internal Medicine, 15^(th) ed. ((Chapter 116) 2001, McGraw Hill, Columbus, Ohio), which are hereby incorporated by reference.

The invention also provides a method of treating abnormalities in clotting due to a defect in the interaction between GPIb alpha and the A1 domain of VWF as occurs in certain types of von Willebrand Disease (VWD), where the method entails administering to the subject an effective amount of a compound that promotes platelet adhesion in the subject, wherein the compound abbreviates off-rate (k_(off)) and/or enhances the on-rate binding kinetics, and or strengthens the bond between VWF-A1 and GPIb-alpha by at least two-fold. Thus, administration of the compound increases blood coagulation in the subject, for example, subjects diagnosed with VWD. In one embodiment, VWD is Type 1 or Type 2. In another embodiment, the compound is one identified by the screening methods described above. Coagulation can be measured by a coagulation factor assay, an ex-vivo flow chamber assay, a platelet adhesion [see EXAMPLES section] or those assays listed in Harrison's Principle of Internal Medicine, 15^(th) ed. ((Chapter 116) 2001, McGraw Hill, Columbus, Ohio).

Therapeutic Formulations

Therapeutic compounds according to this invention are formulated in pharmaceutical compositions containing the compound and a pharmaceutically acceptable carrier. The pharmaceutical composition may contain other components so long as the other components do not reduce the effectiveness of the compound according to this invention so much that the therapy is negated. Some other components may have independent therapeutic effects. Pharmaceutically acceptable carriers are well known, and one skilled in the pharmaceutical art can easily select carriers suitable for particular routes of administration (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985).

The pharmaceutical compositions containing any of the compounds of this invention may be administered by a topical, oral, rectal, parenteral (such as subcutaneously, intramuscularly, intravenously, intraperitoneally, intrapleurally, intravesicularly or intrathecally), or nasal route, as compelled by the choice of drug and disease. One skilled in the pharmaceutical art can discern the optimal route of administration.

These compounds may also be applied topically or locally, in liposomes, solutions, gels, ointments, biodegradable microcapsules, or impregnated bandages. Compositions or dosage forms for topical application may include suspensions, dusting powder, solutions, lotions, suppositories, sprays, aerosols, biodegradable polymers, ointments, creams, gels, impregnated bandages and dressings, liposomes, and artificial skin.

Pharmaceutical carriers utilized by one skilled in the art which make up the foregoing compositions include petrolatum, polyethylene glycol, alginates, carboxymethylcellulose, methylcellulose, agarose, pectins, gelatins, collagen, vegetable oils, phospholipids, stearic acid, stearyl alcohol, polysorbate, mineral oils, polylactate, polyglycolate, polyanhydrides, polyvinylpyrrolidone, and the like.

Therapy dose and duration will depend on a variety of factors, such as the disease type, patient age, therapeutic index of the drugs, patient weight, and tolerance of toxicity. Initial dose levels will be selected based on their ability to achieve ambient concentrations shown to be effective in in vitro models (for example, a dose level used to determine therapeutic index), in vivo models, and in clinical trials. The skilled clinician using standard pharmacological approaches can determine the dose of a particular drug and duration of therapy for a particular patient in view of the above stated factors. The response to treatment can be monitored by analysis of body fluid or blood levels of the compound and the skilled clinician will adjust the dose and duration of therapy based on the response to treatment revealed by these measurements.

EXAMPLES

A number of Examples are provided below to facilitate a more complete understanding of the present invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only.

Biophysical and molecular approaches are essential for understanding the structure:function relationship between a receptor and its ligand. Thus, the ability to study such interactions in an appropriate physiological and/or pathological setting is desirable. To do so, one requires an animal model that is amenable to genetic manipulation and has receptor-ligand interactions that closely resemble those found in humans. Thrombosis models in hamsters and guinea pigs have proven useful in pharmacological studies, but a mouse model would prove to be more beneficial based on the ability to insert or delete genes of interest, accessibility of tissues for study, and cost and ease of handling (49, 50). Regarding GPIb alpha-VWF interactions, two groups have significantly advanced the understanding of the importance of these interactions in mediating thrombosis by generating mice deficient in these proteins (51, 52). Yet, no information regarding the role of the biophysical properties of the GPIb alpha-VWF-A 1 in regulating the processes of thrombosis and hemostasis will be obtained. Thus, the next logical approach is to generate animals with mutations within the VWF-A1 domain that change its kinetic properties in a desired manner and to correlate these biophysical alterations with the ability of these mice to maintain adequate hemostasis and to develop thrombi in response to vascular injury. Such information will be useful in designing therapies that reduce or enhance these processes.

Example 1 VWF Characterization

VWF Microsphere Studies

The association and dissociation kinetics of the GPIb alpha-VWF-A1 bond and the impact of fluid shear and particle size on these parameters can be determined by measuring the frequency and duration of transient adhesive events, known as transient tethers, that represent the smallest unit of interaction observable in a parallel-platelet flow chamber.

Production of recombinant VWF-A1 protein and coating of microspheres. The generation of recombinant VWF-A1 protein (residues 1238 to 1472 of the mature, recombinant VWF) and its subsequent coupling to microspheres is performed as previously described (Doggett, T. A. et al. (2002) Biophys. J. 83, 194-205). Proper size, purity, and disulfide bonding of all proteins is assessed by Coomasie-blue staining of SDS-PAGE gels run under reducing and non-reducing conditions. Mass spectrometry is also employed to evaluate size and disulfide bonding pattern.

The resulting recombinant proteins are bound to polystyrene microspheres (goat anti-mouse IgG (FC); Bangs Lab, Inc., Fishers, Ind.) that were initially coated with a saturating concentration of mouse anti-6-HIS antibody as previously described in our publications. We have found this coating method to be superior to direct covalent coupling of the VWF-A1 to the beads as it prevents significant loss in protein function. Estimation of the amount of VWF-A1 bound to the beads is determined using a monoclonal antibody generated in our lab against the human and murine A1 domains, mAb AMD-1 and mAb AMD-2, respectively, and a calibrated microbead system (Quantum Simply Cellular; Flow Cytometry Standards Corp., San Juan, P.R.) following the manufacturer's instructions.

Laminar flow assays. In flow assays involving protein-coated microspheres, human or murine platelets purified by gel filtration are incubated with 10 mM sodium azide (NaN₃), 50 ng/ml prostaglandin E₁, and 10 μm indomethacin (Sigma Immunochemicals, St. Louis, Mo.) to reduce the possibility of activation and potential alterations in expression and/or distribution of GPIb alpha on their surface. Platelets are subsequently allowed to settle in stasis on Fab fragments of monoclonal antibodies that recognize either human (i.e., mAb 7E3) or murine (i.e., mAb NAD-1) alpha IIb/β₃ in order to form a reactive substrate. The use of platelets in lieu of recombinant proteins or transfected cells as the immobilized substrate enables evaluation of GPIb alpha in its native form (i.e. correct orientation and proper post-translational modification). Platelet coverage of <10% will be bound in this manner can remain relatively unactivated for up to 30 min as evident by morphology on light microscopic examination (FIG. 7A) and lack of expression of P-selectin by fluorescence microscopy (FIG. 7B).

To reduce the possibility of multiple bond formation that would result in a prolongation in interaction times between the beads and immobilized platelets, the lowest site densities of VWF-A1 capable of supporting these brief interactions is used, a value we found to correspond to ˜30 molecules μm². At this site density, we have shown that the formation of transient tethers between this receptor-ligand pair has distribution of bond lifetimes that obey first order dissociation kinetics. The duration of these interactions are measured by recording images from a Nikon X60 DIC objective (oil immersion) viewed at a frame rate of 235 fps (Speed Vision Technologies, San Diego, Calif.) and subjected to wall shear stresses (WSS) ranging from 0.5 to 3.0 dyn cm⁻². The cellular off-rates are determined by plotting the natural log of the number of VWF-A1 coated microspheres that interacted as a function of time after the initiation of tethering, the slope of the line=−k_(off) (s⁻¹) which is the inverse of the bond lifetime. The force acting on the tether bond was calculated from force balance equations as stated above and k_(off) plotted as a function of these forces. An example of the measurement of the duration of a transient tether and estimation of off-rates as a function of WSS is demonstrated below for WT human VWF-A1 (FIG. 8, A-C). To demonstrate that our method for surface immobilization of platelets does not result in an alteration in the kinetics of the GPIb alpha-VWF-A1 tether bond, resting platelets were first fixed in paraformaldehyde prior to immobilization. As these platelets cannot activate, the kinetics should be reflective of GPIb alpha in the resting state. Indeed, analysis of the k_(off) for this interaction using fixed platelets was identical to that observed for platelets treated with metabolic inhibitors (FIG. 8D).

The Structure-Function of Murine VWF-A1

To determine the structure and function of murine VWF-A1, its adhesive interactions with murine and human GPIb alpha, and whether the kinetics of this interaction mimic those reported in studies of its human counterpart, the domain was initially cloned by PCR from purified mouse genomic DNA. For the purpose of generating a mouse with a genetically modified VWF-A1 domain, a100-kb P1 clone was obtained from screening a 129/Svj DNA genomic library (Genomic Systems, St. Louis, Mo.) by polymerase chain reaction (PCR) using primers directed against a 200 bp region of exon 28. Sequence analysis of flanking regions (10 kb in size) as well as the A1 domain itself was performed and compared to those obtained from a BLAST search to confirm the fidelity of the clone. The deduced single-letter amino acid sequence of mouse VWF-A1 domain (M VWF) is shown compared to its human counterpart (H VWF) and encompasses amino acids 1260 to 1480 (FIG. 9). The locations of cysteines forming the loop structure are numbered (1272 and 1458) and differences in residues are highlighted in red. Conversion of the arginine (R) in the mouse A1 domain to histidine (H) as found in its human counterpart (blue χ) has been shown to enable mouse VWF to bind human platelets and simultaneously reduce the binding of mouse platelets. Locations of some, but not all, mutations known to affect human VWF-A1 function are also depicted.

Although the amino acid sequence homology is ˜85%, and preliminary studies suggest that functional differences do exist between human and murine VWF-A1 domains. In a Ristocetin-induced platelet aggregation assays, platelet GPIb alpha binding to wild type human VWF or mouse VWF was analyzed in the absence or presence of ristocetin as described by Inbal, et al. (1993, Thromb. Haemost., 70:1058-1062). In this method, platelet rich plasma (PRP) is placed in a clear cuvette containing a stir bar and inserted into the aggregometer. Platelet aggregation is induced by the addition of ristocetin. In this ristocetin-induced platelet aggregation assay (RIPA), we observe that concentrations of this modulator that are known to cause agglutination of human platelets (˜1.0 mg/ml) had no such effect using murine PRP (FIG. 10B). In fact, only at concentrations of ≧2.5 mg/ml was there any evidence of murine platelet aggregation observed (˜30%, FIG. 10C). In comparison, incubation of murine PRP with thrombin resulted in >90% platelet aggregation (FIG. 10A).

To better evaluate the above interactions and to compare functional relationships between human and murine VWF with GPIb alpha, VWF from human and mouse plasma was purified and its ability to mediate platelet adhesion in flow was determined. Multimer gel analysis did not reveal any differences between the two species, especially with regard to high molecular weight components (FIG. 11A). Moreover, surface-immobilized murine VWF could support adhesion of syngeneic platelets (1×10⁸/ml) at a shear rate encountered in the arterial circulation (1600 s⁻¹) as observed for the human plasma protein (FIG. 11B). In contrast, murine VWF did not support significant interactions with human platelets and vice versa. These results suggest that functional and possibly significant structural differences do exist between the A1 domains of murine and human VWF as primary attachment of platelets at this wall shear rate is dependent on its function. Thus, generation of a recombinant murine VWF-A1 domain is required to fully evaluate similarities and/or differences from its human counterpart.

Recombinant protein was expressed using a bacterial expression vector under the control of an inducible promoter (pQE9, Qiagen). Insertion of the murine fragment containing the majority of the VWF A1-domain (encoding for amino acids 1233 to 1471) into pQE9 produces an amino-terminal fusion protein containing 10 amino acids (including 6× histidine) contributed by the vector. After induction, inclusion bodies were harvested, washed, and solubilized according to previously published methods (32). The solubilized protein was diluted 40-fold in 50 mM Tris-HCl, 500 mM NaCl, 0.2% Tween 20, pH 7.8 and initially purified over a Ni²⁺-chelated Sepharose (Pharmacia) column. To increase the yield of functional protein, the material purified from the Ni²⁺ column was absorbed to and eluted from a Heparin-Sepharose column (Amersham Pharmacia Biotech).

The highly purified protein was dialyzed against 25 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.8. SDS-PAGE analysis revealed a prominent protein band of ˜34,000 Da under non-reducing conditions (FIG. 12). The overall yield of protein obtained using the purification methods described above is ˜2 mg/l of bacterial cells.

The protein was subsequently used in a series of in vitro flow chamber assays to assess function. Washed human or murine platelets (5×10⁷/ml) were infused through a parallel plate flow chamber containing glass cover slips coated with either human VWF-A1 or mouse VWF-A1 protein (100 μg/ml final concentration) at a shear rate of 800 s⁻¹. After 5 min of continuous flow, adherent platelets were quantified.

As shown in FIG. 13A, mouse VWF-A1 protein supported platelet adhesion as efficiently as its human counterpart under physiological flow conditions. To demonstrate the importance of the single disulfide bond formed by C1272 and C1458, reduced (DTT) and alkylated (iodoacetamide) mouse VWF-A1 was prepared and tested in flow. Reduction and alkylation of the protein abrogated attachment of murine platelets in flow. In addition, the limited ability of the native form of the protein to mediate adhesion of human platelets and lack of interaction between human VWF-A1 and mouse platelets suggests that structural/conformational differences exist between the species. However, this does not preclude the study of GPIb alpha-VWF-A1 interactions in mice as both proteins must share common kinetic attributes as they support rapid attachment and translocation of platelets to a similar degree under physiological flow conditions (FIG. 13B).

To demonstrate that the presence of the N-terminus His tag does not appear to affect the function of the recombinant protein, we compared the ability of a tagged vs. a non-tagged M VWF-A1 to support mouse platelet adhesion in flow. In the case of the latter, the murine A1 fragment was inserted into pET-11b (Stratagene) and purified as previously described (53). The purified bacterial non-His tag protein was analyzed by SDS-PAGE (12.5%) and found to migrate in an analogous manner to its tagged counterpart under non-reducing and reducing conditions (FIG. 14A). In addition, no differences were observed in the number of platelets that adhered to and translocated on either protein (449±53 platelets/mm² His-tag vs. 423±17 platelets/mm² non-His tag) at a shear rate of 800 s⁻¹ (FIG. 14B).

Characterization of the M VWF-A1 Domain.

The A1 domains of human and mouse serve an identical purpose: to mediate primary attachment and translocation of platelets in flow. The crystal structure of the mouse A1 domain was solved using recombinant proteins (Fukuda, K., et al., (2005) Nat. Struct. Mol. Biol. 12:152-159). The main chain schematic of this domain, with β-strands (arrows) and helices (coils), is shown in FIG. 15A. The model was built from residues 1270 to 1463 of the murine VWF-A1 crystal. The two cysteines involved in the disulfide bridge are shown as yellow spheres (involving residues 1272 and 1458). The mouse and human A1 domains appear to overlap very closely, which suggests that only minor structural differences may account for the preferential binding of platelets from mice or man to their respective VWF-A1 proteins (FIG. 15B). In fact the β-sheets of both species are identical within experimental error (a root mean square difference of 0.33 Å for Cα atoms). Thus, minor differences in residues, but not structure, most probably account for the inability of human platelets to interact with mouse VWF-A1 and vice versa.

Support for this hypothesis is provided by mutagenesis studies. By analyzing the data obtained from the crystal structure of the murine VWF-A1 domain, we have identified several residues that may participate in interactions with GPIb alpha (FIG. 15C). Residue 1326 was initially chosen for study and was mutated to the corresponding amino acid at the identical location in its human counterpart (from Arg to His). Subsequently, the ability of murine and human platelets to interact with this mutant protein substrate was evaluated at a wall shear rate of 800 s⁻¹. Incorporation of a histidine for arginine at position 1326 in murine VWF-A1 reduced murine platelet adhesion by ˜5-fold and increased translocation velocities of cells by ˜7-fold as compared to the WT mouse protein (FIGS. 13 and 16). Interestingly, human platelet interactions with the mutated murine protein were comparable to that of WT human VWF-A1. Conversely, substitution of Arg for His in the human VWF-A1 protein resulted in an increased ability of murine platelets to attach and translocate in a manner similar to that observed for WT murine VWF-A1. These studies support the hypothesis that from a structural and functional standpoint, mouse and human VWF-A1 are very similar.

Thus, all that remains is to demonstrate that the kinetics of the interaction between the murine GPIb alpha and murine VWF-A1 are similar to its human counterpart and that mutations in man that cause functional alterations in platelet adhesion with VWF have the identical impact on the biophysical properties of the murine receptor-ligand pair.

The Kinetics of Murine VWF-A1

To determine whether the kinetics of the murine GPIb alpha interactions with the murine VWF-A1 domain is similar to that of the human receptor-ligand pair, we measured the dissociation of transient tethering events using VWF-A1 coated beads (7 μm diameter) interacting with surface-immobilized platelets. The use of beads with one uniform size and shape permits determination of the relationship between wall shear stress and the force directly acting on the GPIb alpha-VWF-A1 tether bond (F_(b)), a parameter difficult to estimate for discoid-shaped objects such as platelets. A coating concentration of VWF-A1 was chosen (5 μg/ml corresponding to 30 molecules/μm²) that supported tether bond formation at wall shear stresses ranging from 0.5 to 3 dyn cm⁻². Estimation of the site density of murine VWF-A1 on beads was performed using a monoclonal antibody generated in our laboratory designated as AMD-2. This antibody was made by immunizing Fischer 344 rats (3-4 months old) with recombinant WT protein. Following several injections of murine VWF-A1, serum was collected and screened by ELISA for anti-VWF-A1 antibodies. Spleens from animals with the highest antibody titers were harvested and splenocytes fused with Sp2/0 mouse myeloma cells (54).

Supernatants of hybridomas were screened for reactivity to mouse VWF-A1 by ELISA (FIG. 17). Pre-immune rat serum was used as control. Monoclonal antibodies (Mabs) to murine VWF-A1 not only reacted with WT and mutant proteins (1324G>S) but also recognized native VWF purified from mouse plasma. Antibodies are currently being tested for function blocking capabilities to use in both in vitro and in vivo assays. Antibodies will also be used for epitope mapping.

Analysis of the distribution of interactions times between human or murine VWF-A1 coated beads and their respective platelet substrates, as measured by high temporal resolution videomicroscopy, indicate that >95% of all transient tether bonds events fit a straight line, the regressed slope of which corresponded to a single k_(off) (FIGS. 18A-C). Notably, the cellular off-rates of these quantal units of adhesion for the WT human and mouse proteins (FIGS. 18A and B) were quite similar, but were significantly higher than those observed for the murine VWF-A1 containing the type 2B mutation I1309V (1309I>V) (FIG. 18C). This is consistent with previous results obtained using the same mutation in the human protein (Table 1).

TABLE 1 SINGLE H1326R* G1330E* R1287M* Q1391P* A1350T* S1370G* D1333A*

Based on these preliminary results, it appears that the dissociation kinetics of murine GPIb alpha interactions with murine VWF-A1 are nearly identical to its human counterpart and that type 2B mutations also prolongs the bond lifetime of this interaction as seen in man. A complete biophysical analysis is underway and in order to determine values for the intrinsic k_(off) and susceptibility of the bond to force drive dissociation as performed previously for its human counterpart.

Example 2 VWF-A1 Mutagenesis

Preliminary results indicate that minor differences may exist between murine and human VWF that would preclude one from studying human platelet behavior in a mouse model of thrombosis. However, our findings that the estimated off-rate values and structure of these domains are similar suggest that one can investigate the role of the biophysical properties of the GPIb alpha-VWF-A1 bond in regulating platelet-VWF interactions in vivo using a mouse model. However, neither a delineation of the binding region for GPIb alpha within the murine VWF-A1 domain nor determination of the impact of mutations on the kinetics of this interaction has been performed to date. Thus, both murine and human A1 crystal structures can be exploited to 1) identify candidate residues involved in the binding site for murine GPIb alpha and to determine their impact on the kinetic properties of this receptor-ligand pair, 2) identify residues that confer species specificity, and 3) ascertain whether insertion of known point mutations that cause type 2M and 2B VWD in man alter the kinetic properties of the murine A1 domain in a similar manner. Critical residues can be classified in terms of their impact on the cellular association and dissociation rate constants. Information obtained from these studies can be used to generate mice with mutant A1 domains in order to establish the degree in alteration in the kinetics of the GPIb alpha-VWF-A1 bond that is necessary to perturb hemostasis.

Similar critical structural elements exist in murine A1 domain to those identified in its human counter-part that contribute to the biophysical properties of the bond formed with GPIb alpha. Thus, to identify structural elements within the murine VWF-A1 domain that impact on the kinetics of interaction with GPIb alpha, the hypothesis that only minor structural alterations in this domain are responsible for its reduced ability to support interactions with GPIb alpha receptor on human platelets will be tested.

Site-Specific Mutagenesis of Murine VWF-A1 Domain

Site-specific mutagenesis of murine VWF-A1 domain will be performed to define residues that contribute to GPIb alpha binding as well as those in that regulate this interaction. Studies will initially focus on amino acids that differ between human and mouse A1 that lie within the vicinity of the proposed GPIb alpha binding pocket.

To better define residues within murine VWF-A1 that are critical for binding of GPIb alpha on mouse platelets, mutations into VWF-A1 cDNA using a PCR-based strategy will be introduced and the resulting DNA will be sequenced to confirm the presence of the desired mutation(s). Mutations will be based on the murine A1 crystal structure and amino acid substitutions known to affect human VWF function such as those associated with Type 2M or 2B vWD (Tables 2-4). Several surface exposed residues have been identified within the murine A1 domain likely to participate in GPIb alpha binding. These are non-conserved residues in comparison to the human domain. Thus, we will convert these residues at first singly (then doubly and triply), into the murine VWF-A1 to those found in human VWF-A1*. Residues are chosen based on surface-exposure on the front and upper surfaces of the domain as understood by modeling and crystal structure analysis.

TABLE 2 SINGLE R1326 H* E1350G* M1287R* P 1391 Q* T1350A* G1370S* A1333D* DOUBLE R 1287 M* + Q 1391 P* TRIPLE H 1326 R* + G 1350 E* + A1333D*

TABLE 3 SINGLE S1289R* D1323R* K1348E* R1392E* Residues that perturb but do not abrogate platelet binding in the human VWF-A1 protein (Table 3, FIG. 5B).

TABLE 4 TYPE 2M G1324S* Q1367R* I1369F* I1425F* TYPE 2B R1306L* I1309V* V1316M* R1341L* Residues chosen based on their ability to abrogate or enhance interactions between human VWF and GPIb alpha (Table 4).

Type 2B mutations will also be combined with those that dramatically shorten the bond lifetime to determine (increase k_(off)) if these function-enhancing mutations can restore adhesion to that observed for WT VWF-A1.

Laminar flow assays will be performed to assess the impact of various mutations on platelet adhesion as well as the degree in alteration in the kinetic properties of the GPIb alpha-VWF-A1 bond.

Murine platelets will be purified as described above and stored in Tyrode's buffer containing 0.25% BSA, pH 7.4. For studies requiring human platelets, blood will be collected by venopuncture from healthy donors and cells obtained from centrifugation of PRP. All platelets will be used within 2 hours of purification.

To evaluate the impact of mutations on platelet adhesion, both human and murine platelets are perfused over high concentrations of murine VWF-A1 proteins (100 μg per ml) absorbed to glass cover slips in a parallel plate flow chamber at wall shear rates ranging from 20 to 1600 s⁻¹. An enzyme-linked immunosorbent assay is utilized to ensure that an equivalent amount of recombinant WT or mutant protein is immobilized. The number of platelets that attach per unit area per min and their velocity of forward motion (μm/s), termed translocation, is recorded on Hi-8 videotape using an inverted Nikon microscope with a plan 10× or 40× objective, respectively. In addition, whether the incorporated mutations alter the requirement for a critical level of hydrodynamic flow, termed the shear threshold phenomenon, to support interactions of platelets with the reactive substrate can be determined. It has been previously demonstrated that human attachment to immobilized VWF-A1 requires a minimum of >85 s⁻¹ of WSR to initiate and sustain this interaction. This phenomenon has also been well described for selectin-dependent adhesion and is believed to rely on a balance between the number of times a receptor encounters it ligand over a defined period of time and the rate at which a bond can form, parameters affected by shear rate, association rate constant, and receptor-ligand concentrations (59, 60). Once attached, however, the bond lifetime influences the velocity at which the cell will move on the reactive substrate in response to shear-induced force (61). Thus, it is likely that several mutations may perturb platelet accumulation on surface-bound VWF-A1 by altering the level of shear flow required to promote platelet attachment as well as the translocation velocities of these cells. For example, it has been shown that the type 2B mutation, Ile1309Val (1309I>V), promotes greater platelet attachment at low shear rates and reduces their translocation velocities as compared to the WT substrate. Similar results were observed upon incorporation of the identical mutation into murine VWF-A1. Demonstration that GPIb alpha on murine platelets is responsible for mediating interactions with recombinant A1 domains can be confirmed by antibody blocking experiments.

Determination of Tethering Frequencies, Translocation Velocities, Detachment Profiles, and Dissociation Rate Constants Using VWF-A1 Coated Microspheres.

To better ascertain the alteration in kinetics associated with the proposed mutations, the tethering frequency, translocation velocities, detachment profile, and off-rates of VWF-A1 coated beads (7 μm diameter) interacting with surface-immobilized platelets can be measured. As stated before, the use of beads with one uniform size and shape will permit determination of the relationship between wall shear stress and the force directly acting on the GPIb alpha-VWF-A1 tether bond (F_(b)), a parameter difficult to estimate for discoid shaped objects such as platelets. Platelet coverage >90% of the glass surface area is used in determining the tethering frequency (on rate driven phenomenon), translocation velocities (correlates with off-rate) and resistance to detachment forces (measure of bond strength) of VWF-A1 coated beads in flow. Recently, we have demonstrated that comparison of cellular on-rates and apparent bond strengths between WT and mutant forms of VWF-A1 can be achieved by limiting the concentration of these molecules as to prevent multiple bond formation, a process that can mimic an enhancement in either of these kinetic parameters. By using a similar strategy, we can determine whether the proposed mutations will alter the apparent on-rate of the GPIb alpha-VWF-A1 bond by evaluating the frequency of transient tethering events between microspheres coated with low site densities of VWF-A1 proteins and a platelet substrate. Results are expressed as the percentage of beads (per 10× field) that paused, but did not translocate, on over a range of wall shear rates that support such interactions (20 to 400 s⁻¹). Tethers per minute are divided by the flux of beads near the wall per minute to obtain the frequency of this adhesive interaction. Only one tethering event per bead is counted during the observation period.

For determining translocation velocities, beads (1×10⁶/ml), coated with a saturating concentration of VWF-A1 protein are infused into the parallel-plate flow chamber at 1.0 dyn cm⁻² and allowed to accumulate for 5 min. Subsequently, the wall shear stress is increased every 10 s to a maximum 36 dyn cm⁻² and the velocities of the beads determined.

For detachment assays, beads (1×10⁶/ml), coated with the minimum but equal amounts of VWF-A1 required to support translocation, are infused into the parallel-plate flow chamber at 1.0 dyn cm⁻² and allowed to accumulate for 5 min. Subsequently, the wall shear stress is increased every 10 s to a maximum 36 dyn cm⁻². The number of beads remaining bound at the end of each incremental increase in wall shear stress is determined and expressed as the percentage of the total number of beads originally bound. Using this strategy we were able to support our claim that type 2B mutations do not strengthen, and in fact may even weaken the interaction between GPIb alpha and VWF-A1 as suggested by the increase in reactive compliance as compared to the native complex (Table 1). In all studies, video images are recorded using a Hi8 VCR (Sony, Boston, Mass.) and analysis of performed using a PC-based image analysis system (Image Pro Plus).

For determining the kinetics of dissociation, we measure the duration of transient tethers between murine VWF-A1 coated microspheres and immobilized murine platelets as described herein. MC simulations are run and estimates of k_(off) fit to the Bell model by standard linear regression to obtain the intrinsic off-rate (k⁰ _(off)) and the reactive compliance σ. Results are compared for all mutations to determine their impact on the these kinetic parameters (i.e.—increase or shorten the bond lifetime (k⁰ _(off)) and/or increase or decrease the susceptibility of the bond to hydrodynamic forces (σ).

These experiments complement recent work on identifying residues in human VWF-A1 domain critical for interacting with the GPIb alpha. Moreover, they allow for delineation of its binding site in murine VWF-A1. We believe this to be important, as this work will be essential for elucidating the role of the biophysical properties of this receptor-ligand pair in regulating platelet-VWF interactions in vivo. Furthermore, it will pave the way for the generation of mice with comparable types of human vWD (i.e. type 2B) and may even permit the study of human platelets in a mouse model of thrombosis.

Although our approach to mapping the GPIb alpha binding site is reasonable based on our previous studies, there is no guarantee that the introduction of mutations will not significantly perturb protein structure and thus function. The ability of murine VWF-A1 specific mAbs to recognize mutant proteins should clarify this matter. Similarly, our proposed gain/loss of function experiments involving swapping of residues between human and mouse VWF-A1 will also prove useful in avoiding this pitfall.

It is important to know whether the regions flanking the mouse A1 domain are important in mediating interactions with GPIb alpha. To this end, we plan to express full-length mouse VWF by inserting it into a mammalian expression vector and transfecting it into COS-7 cells (62). Mutations found to be critical for binding, will be inserted into the full-length construct. As we are in the process of generating the full-length cDNA, we will initially attempt to generate a recombinant protein containing the A1-A2-A3 domains to use in our studies. This will be accomplished using a baculovirus expression system as demonstrated for GPIb alpha.

The Relationship Between the Major and Minor Binding Sites for GPIb Alpha.

The recent results on the structure of GPIb alpha and its complex with VWF-A1 domain has not only confirmed our work as well as others with regard to the major binding site for this platelet receptor, but has shed new light into the mechanism by which type 2B mutations may enhance this critical interaction. As shown in FIG. 6, the concave face of GPIb alpha embraces the A1 domain in two distinct regions. The C-terminal loop of this receptor binds near the top of the domain (major binding site) and the N-terminal region known as the β-finger, at the bottom face (minor binding site) adjacent to the site where type 2B mutations are clustered. Based on these results that type 2B mutations appear to enhance the on-rate (reduced shear rate needed for formation of transient tethers in flow) and prolong the lifetime (5-6 fold) of the interaction between VWF-A1 and GPIb alpha, it is interesting to speculate whether similar alterations in bond kinetics would be observed with type 2B mutations if one interfered with the primary site. For instance, would inclusion of a type 2B with a type 2M mutation reconstitute adhesion, or is some finite interaction time required in the primary binding pocket for GPIb alpha before the effects of these mutations can be observed? These are important questions as they will guide the development of reagents that can either enhance or reduce the interaction between GPIb alpha and VWF-A1.

The type 2B mutation 1309I>V was incorporated into recombinant human VWF-A1 containing either the type 2M mutation Gly1324Ser (1324G>S) that completely abolishes adhesion or the function reducing mutation His1326Arg (1326H>R) and determined the ability of these doubly mutated proteins to support human platelet adhesion in flow. In comparison to WT, a ˜3-fold increase in wall shear rate is required to promote platelet attachment to human VWF-A1 containing Arg at 1326 (FIG. 19A). Incorporation of the type 2B mutation, however, appeared to enhance the on-rate of this interaction as manifested by an increase in platelet binding at lower levels of shear flow, but not to levels observed for the type 2B mutation alone.

To determine whether the 1309 mutation would also prolong the lifetime of the interaction with GPIb alpha, the distribution of interactions times was analyzed between VWF-A1 coated beads and surface-immobilized platelets at a wall shear stress of 1 dyn cm⁻². Remarkably, a 2-fold reduction in k_(off) was noted (from 15.6 to 8.5 s⁻¹) as compared to the single, function-diminishing mutation (FIG. 19B). This value is similar to that of the native receptor-ligand interaction with a k_(off) of 6.5 s⁻¹ under identical flow conditions. By contrast, incorporation of Val for Ile at residue 1309 in an A1 domain containing the type 2M mutation 1324G>S did not reconstitute platelet adhesion. Thus, these results suggest that it is essential for bond formation to occur in the primary GPIb alpha binding site (top face of the A1 domain). Moreover, the region of the A1 domain where type 2B mutations are clustered appears to be critical for stabilizing interactions with GPIb alpha. Similar findings were observed for murine VWF-A1 containing the identical type 2B mutation but with a change in Arg to His at residue 1326. A complete biophysical analysis is underway and in order to determine the full extent of the 1326 mutation on the intrinsic k_(off), the susceptibility of the bond to force drive dissociation, and whether type 2B mutations can restore these parameters to levels obtained for the native receptor-ligand bond. In addition, it would be interesting to determine in vivo whether the enhancement in binding and increase in bond lifetime imparted by the type 2B mutation would correct any perturbation in hemostasis that may occur as a result of impacting on the function of the primary binding site for GPIb alpha.

Example 3 Genetically Modified VWF-A1 Mice

Recent kinetic evaluation of mutations associated with type 2B and platelet-type vWD suggests that the intrinsic properties of the GPIb alpha-VWF-A1 tether bond contribute to the regulation of platelet interactions with VWF. This is also supported by our preliminary studies investigating the impact of botrocetin on the biophysical properties of this receptor-ligand pair. Thus, by using the information obtained in Example 2, mutations can be incorporated into the murine A1 domain of the VWF gene that increase or decrease the intrinsic on- and off-rates by varying degrees in order to truly understand the importance of these kinetic parameters in controlling platelet adhesion. Moreover, the role of the minor binding site, where the majority of type 2B mutations have been identified, can be further delineated by combining such mutations with those that significantly shorten the lifetime of interaction between GPIb alpha and VWF-A1. Results indicate that substitution of the murine residue Arg at position 1326 for His at the same location in the human A1 domain results in a diminished on-rate as manifested by the increased requirement for shear flow to promote attachment and a significant increase in k_(off) (shortening of bond lifetime). Subsequent incorporation of the type 2B mutation 1309I>V into this mutant domain significantly reverses the functional defect in adhesion and returns the off-rate closer to that observed for the WT domain. Similar results have been obtained with murine VWF-A1 in which Arg was replaced by His at residue 1326. Thus, introduction of these two mutations separately and then together into the mouse VWF gene will be the initial focus. This single amino acid substitution enables the mouse VWF-A1 domain to bind platelets at levels equivalent to its human counterpart. Thus, introduction of the two mutations separately (1326R>H or 1309I>V) and then together into the mouse VWF gene has been the target of recent studies. In addition, replacing >90% of the entire mouse A1 domain with its human counterpart is also central to current investigations. This model will enable one to test all potential therapies directed against this human domain in a mouse model of thrombosis.

Generation of mice that incorporate mutations into their A1 domain that significantly shorten the bond lifetime will be present with prolonged bleeding times and will be resistant to thrombus formation, while the additional incorporation of a type 2B mutation will correct these abnormalities by prolonging the tether bond lifetime to that observed for the WT domain. This should allow for sufficient time to form multiple bonds between platelets and VWF deposited at sites of vascular injury. Moreover, mice possessing the 1326R>H mutation should be able to support human platelet adhesion at sites of vascular injury. Thus, it will be demonstrated in vivo that the intrinsic properties of the GPIb alpha-VWF-A1 tether bond are indeed critical for regulating the interactions between platelets and VWF at sites of vascular injury.

By performing a detailed kinetic analysis of mutant VWF-A1 domains prior to the generation of animals with the identical substitutions in amino acids, we have greatly increased the likelihood of altering the interaction between platelets and VWF in a similar manner. We will be able to study the role of the intrinsic properties of the bonds formed between this receptor-ligand pair under complex hemodynamic conditions (i.e. in vivo). The initial choice of the 1326R>H mutation will be of benefit in two ways. First, it allows us to test our hypothesis that a critical interaction time between platelets and VWF is essential to maintaining adequate hemostasis. Secondly, the ability of this mutation to enhance human platelet adhesion will permit us to study their behavior in a mouse model of thrombosis. This is an intriguing concept as it may pave the way to test the impact of various pharmacological agents on human platelet adhesion at sites of vascular injury. For example, a substitution in which a single mutation in murine VWF-A1 (1260 to 1480) can be made in order to achieve human platelet bonding. In addition, a combination of two or more mutations that further perturb the kinetics of the interaction to achieve human platelet binding can also be made. In some instances, the entire mouse A1 domain in the mouse VWF gene with the human A1 domain found in the human VWF gene can be replaced.

Generation and Characterization of Mice Expressing a Mutant VWF-A1 Domain

Mutant mouse. As mentioned previously, a 100-kb P1 clone containing the majority of the VWF gene (Genomic Systems, St. Louis, Mo.) was obtained. Digestion with Bam H1 resulted in a ˜5.3 kb fragment containing part of intron 28 (including the splice sites), all of exon 28 and part of intron 29 which was the inserted into the pSP72 vector (Promega, WI). This was subsequently digested with Bam H1 and Eco R5 to yield a 2.9 kb (including exon 28) and a 2.4 kb fragment, designated Arm 1 and 2, respectively, both of which were subcloned back into pSP72 vector. This facilitated site-directed mutagenesis of the A1 domain contained within Arm 1. In addition, the 3′ end of Arm 1 was extended 2 kb by PCR. Subsequently, Arms 1 and 2 were inserted into a lox P-targeting vector as shown below (FIG. 20). The fidelity of three constructs containing either the 1309I>V or 1326R>H substitutions or both mutations was confirmed by sequence analysis.

R1 embryonic stem cells derived from a 129/Sv X 129/Sv-CP F1 3.5-day blastocysts were electroporated with 25 μg of linearized targeting construct and selected in both G418 (26 μmol/L) and gancyclovir (0.2 μmol/L). Genomic DNA from resistant clones were digested with EcoR1 or KpnI, and analyzed by Southern blot hybridization with probe “a” or “b”, respectively, to determine if the construct was appropriately targeted (FIG. 20B). Targeting both the type 2B (Ile1309Val or 1309I>V) mutation and the Arg1326His (1326R>H) mutant constructs, have been successful. In a second step, embryonic stem cell clones that had undergone homologous recombination were transfected with 25 μg of Cre-recombinase-expressing plasmid and selected for G418. Clones in which the neo-cassette was deleted were identified by PCR and injected into C57BL/6 blastocysts (The Siteman Cancer Center Core Facility, Washington University). Male chimeric mice were bred to C57BL/6 Cre-recombinase (+) females to obtain heterozygous animals. Heterozygous mice lacking the neocassette, but containing the 1326R>H mutation, were interbred to obtain wild-type, heterozygous, and homozygous animals. Animals were identified by both Southern analysis (FIG. 21) and by PCR of the A1 domain (FIG. 22; red boxed area denotes the conversion of Arg to His).

Determination of the multimeric composition of murine VWF. For platelet counts, whole blood will be collected into heparinized tubes and 100 μl volumes will be analyzed on a Hemavet (CBC Tech, Oxford, Conn., USA) Coulter Counter. The multimeric structure of murine VWF will be assayed by using the Pharmacia Phast Gel System (Pharmacia LKB Biotechnology). Briefly, samples diluted in 10 mmol/L Tris/HCl, 1 mmol/L EDTA, 2% SDS, 8 mol/L urea, and 0.05% bromophenol blue, pH 8.0, will be applied to a 1.7% agarose gel (LE, Seakem, FMC Bioproducts) in 0.5 mol/L Tris/HCl, pH 8.8, and 0.1% SDS with a stacking gel consisting of 0.8% agarose (HGT, Seakem) in 0.125 mol/L Tris/HCl, pH 6.8, and 0.1% SDS. After electrophoresis the protein will be transferred to a polyvinylidene fluoride membrane (Immobilon P, Millipore) by diffusion blotting for 1 hour at 60° C. The membrane will be blocked with 5% nonfat dry milk protein solution for 1 hour at room temperature. After washing with PBS/T, pH 7.4, the blot will be incubated with a polyclonal antibody raised in rabbits against murine VWF at a dilution of 1:500, washed, and incubated with a goat anti-rabbit horseradish peroxidase (Sigma) diluted 1:2000 in PBS/T. After three washes with PBS/T, the membrane will be incubated with the substrate solution (25 mg 3,3′-diaminobenzidine tetrahydrochloride (Sigma) in 50 mL PBS with 10 μL 30% H₂O₂). The enzyme reaction will be stopped by washing the membrane with distilled water.

To demonstrate that conversion of Arg to His in the mouse A1 domain at position 1326 did not alter plasma protein levels of VWF nor its ability to form multimers, we performed an ELISA to detect mouse VWF in plasma obtained from WT and homozygous animals (FIG. 23). As shown in FIG. 23, plasma levels of VWF from homozygous mice (KI) were comparable to WT at all dilutions tested. Moreover, multimer gel analysis of plasma VWF revealed an identical banding pattern between mouse and human VWF. Incorporation of His at position 1326 in the mouse A1 domain had no effect on multimerization of VWF (FIG. 24). Thus, we are the first to successfully introduce a point mutation into mouse VWF A1 domain.

Bleeding time for human platelet-induced hemostasis: This assay provides an indirect measure of the ability of platelets and VWF to support hemostasis by interacting with the injured vessel wall. It also indirectly determines the function of multiple receptors and ligands on platelets that are required to form a hemostatic plug. That said, it provides direct evidence that the bleeding defect in our animals can be corrected by the administration of human platelets (FIG. 12). It is performed by immersing the severed tip (10 mm) of the animal's tail in isotonic saline at 37° C. and monitoring the length of time required for bleeding to cease. Homozygous mutant mice will be infused with an equal volume of either saline or purified human platelets. Platelet specific antibodies or drugs will be administered as described above and their ability to prolong bleeding time evaluated. All experiments will be stopped at 10 min by cauterizing the tail (51).

Although homozygous mice bearing the 1326R>H mutation are viable, they demonstrate a bleeding phenotype similar to that of animals deficient in VWF (KO) only when 10 mm of the tail is cut (FIG. 25 and FIG. 35B). Over 90% of these mice bled for a minimum of 8 minutes (end point) in contrast to 3.5 minutes for WT animals as measured by severing 1 cm of their tail. Moreover, thrombus formation induced by perfusion of whole blood from mutant mice over surface-immobilized collagen in vitro was reduced by ˜80% as compared to WT controls (FIG. 26). To further characterize the bleeding phenotype depicted in FIG. 35, standard techniques known in the art were employed, which involves removal of a minimal amount of the animal's tail (˜5 mm). A slight, but statistically significant (P<0.01), increase in bleeding was observed in the homozygous 1326R>H mutant mice as compared to WT littermates, but not to the extent of the VWF knockout mouse (a mouse model that bleeds profusely due to complete lack of this plasma glycoprotein). In the homozygous 1326R>H mutant mouse, a larger cut (˜10 mm) is needed in order to see a bleeding phenotype comparable to the VWF deficient mouse. Since these mice continue to bleed, the experiment is stopped at 8 min in order to prevent death. This phenotype observed in the 1326R>H mutant mouse is similar to the bleeding observed in human patients who have type 2M VWF disease. As not all type 2M mutations result in a complete loss of interaction between GPIb alpha and VWF-A1, but resemble the adhesion defects outlined for the 1326R>H mutant mouse, this genetically modified mouse model will be useful for directing therapies aimed at patients that have a partial but not complete defect in binding between GPIb alpha and VWF-A1.

Platelet Adhesion Studies in Mice Expressing a Mutant VWF-A1 Domain

One goal of the work is generate mice with mutant A1 domains that alter the kinetics of its interactions with GPIb alpha on mouse platelets. The first mutation we chose to introduce was the substitution of histidine for arginine at position 1326. This mutation was chosen based on our crystal structure analysis of the mouse and human A1 domains, which suggested that the location of this amino acid is central to GPIb alpha binding. Mice bearing this mutation are viable and demonstrate a bleeding phenotype, albeit not as severe as those lacking VWF (VWF KO) (FIG. 35). This was not unexpected as VWF is still present, but has a reduced ability to interact with platelets at high shear rates (>1600 s⁻¹). FIG. 27 demonstrates reduced thrombus formation that occurs when whole blood from these knock-in animals is perfused over collagen-coated cover slips at a shear rate of 1600 s⁻¹. Results thus far indicate a 70% reduction in thrombi formed on collagen as compared to WT controls.

To demonstrate that the 1326R>H mutant in the A1 domain of mouse VWF (wherein the mutant VWF-A1 domain comprises SEQ ID NO: 5) is far superior to promoting interactions with human platelets under physiologic flow conditions, anticoagulated human blood was infused over surface-immobilized WT or mutant mouse plasma VWF at 1600 s⁻¹ (FIG. 28). Results indicate that the mutant form of mouse VWF can support human platelet attachment to levels observed for its human counterpart, thus making it an ideal system to evaluate human platelet behavior and the impact of novel anti-thrombotic drugs in an animal model. Moreover, this observation was not limited to ex vivo studies, as homozygous mutant mice infused with human (FIG. 29A) but not mouse platelets (FIG. 29B) were able to generate an arterial thrombus that occludes the vessel lumen in response to laser-induced vascular injury as depicted by intravital microscopy (transmitted light). Preliminary results indicate that mouse and human A1 domains are structurally similar and serve an identical functional role in the initiation of thrombus formation. Moreover, the ability of the “humanized” mouse A1 domain to support human platelet adhesion to the same degree as its human VWF-A1 counterpart ex-vivo, as well as its preferential binding of human platelets in vivo (FIG. 29A), suggests that our animal model will be an ideal system for preclinical screening of therapies directed at limiting the interactions between GPIb alpha and the VWF-A1 domain. Moreover, as both hemostasis and thrombosis also rely other key adhesion receptors on human platelets, such as those that interact with collagen OR fibrinogen FIG. 1B), this model can also be used for testing therapies directed against other human platelet receptors and ligands critical for these processes.

To assess the biological significance of this finding in terms of its effect on hemostasis, homozygous mutant animals received an infusion of blood-banked human platelets and bleeding time was subsequently measured by severing 1 cm of their tail. Average bleeding time for mice that received human platelets was ˜3 minutes vs. 10 minutes (end point) for animals given an intravenous infusion of a physiological buffered saline solution (FIG. 30). Results indicate that not only can the mutant form of mouse VWF support human platelet adhesion both ex-vivo and in vivo assays, but it can also perform its biological function; supporting hemostasis in the context of vascular injury.

Evaluation of platelet-VWF behavior in flow. Blood will be collected by cardiac puncture from anesthetized mice and thrombin-mediated activation prevented by the addition of hirudin (160 U/ml, Sigma) (68). Platelet adhesion to a glass cover slip coated with 100 μg/ml of equine tendon collagen (Helena Laboratories, Beaumont, Tex.) will be assessed in a parallel-plate flow chamber apparatus. Whole blood will be infused through the chamber at a wall shear rate of 1600 s⁻¹ for 3 minutes. As platelet adhesion under these homodynamic conditions requires VWF deposition and subsequent interactions between its A1 domain and GPIb alpha, the extent of platelet coverage should provide a gross estimate of the degree in impairment between this receptor-ligand pair. In addition, plasma VWF will be purified from these animals to evaluate platelet attachment to this immobilized substrate in flow. The surface area covered by adherent platelets at the end of each experiment will be determined (Image Pro Plus software) and expressed as a percentage of platelet coverage using blood from WT littermates. To better isolate GPIb alpha-VWF A1 interactions, identical experiments can be performed using platelets isolated from alphaIIb beta 3 deficient animals and reconstituting them in platelet poor plasma from our mutant A1 knock-in mice.

Evaluation of platelet-VWF behavior in vivo. In addition to the proposed in vitro work, platelet-VWF interactions in vivo will also be studied using intravital microscopy (Falati et al. (2002) Nature Medicine 8(10): 1175-80). This is accomplished by using a murine model of thrombosis that involves laser-induced injury to micro-vessels contained within the mouse cremaster muscle. The surgical preparation of animals, insertion of lines for administration of cells and anesthesia, will be performed as previously described (69). Human platelets will be collected and prepared, fluorescently labeled, perfused into a mouse model (such as the transgenic mouse of the current invention) via an intravenous injection (Pozgajova et al., (2006) Blood 108(2):510-4).

Surgical preparation of animals: Insertion of lines for administration of cells and anesthesia. Briefly, the skin covering the scrotum will be incised and the intact cremaster muscle dissected free from the connections to the subcutis. The mouse will be placed on a custom-built plexiglass board, and the exposed muscle positioned on a heated circular glass coverslip (25 mm) for viewing. The muscle will be slit along the ventral surface (using a thermal cautery), the testis excised, and the muscle spread across the coverslip with attached sutures (6/0 silk) (FIG. 31). The cremaster muscle will be kept continuously moistened by superfusion throughout the experiment with sterile, bicarbonate-buffered (pH 7.4), saline solution (37° C.) that is pregased with a 5% CO₂, 95% N₂ mixture for O₂ depletion. All parts of the setup in contact with the superfusion buffer will be presoaked with 1% Etoxaclean (Sigma Chemical Co., St. Louis, Mo.) overnight followed by extensive rinsing in 70% ethanol and endotoxin-free distilled water. The number of mice used for these experiments will be kept to the minimum necessary to establish statistically significant observations. Anesthetized animals will be euthanized after each experiment by CO₂ inhalation.

Vascular trauma will be generated as follows: The segment of an arteriole will be visualized and recorded as “pre-injury”. Subsequently, endothelial damage will be induced via a pulsed nitrogen dye laser at 440 nm applied through the microscope objective using the Micropoint laser system (Photonics Instruments, St. Charles, Ill.). The duration of exposure of the endothelium to the laser light will be varied to produce either a mild injury that supports the formation of a platelet monolayer or significant injury resulting in thrombus formation. The region of interest will then be videotaped and analyzed as described below.

For example, vascular damage can subsequently be induced in arterioles contained within the cremaster muscle of mice by either 1) pulsed nitrogen dye laser applied through the objective of an intravital microscope (FIG. 32) or 2) standard application of a ferric chloride solution (Furie et al. J. Clin. Invest. 2005; 115:3355). The latter method has the advantage of exposing significant more subendothelial collagen, which will be beneficial for testing the role of the collagen receptors α2β1 in thrombus formation.

For studies analyzing the dynamic interactions between individual platelets and the injured vessel wall (attachment, translocation, and sticking), cells purified from genetically altered mice will be labeled ex-vivo with a derivative of carboxyfluorescein (BCECF, Molecular Probes) (Diacovo et al. Science. 1996). A human thrombus generated in the mutant mouse can also be visualized by this technique, thus allowing to distinguish human platelets from endogenous circulating mouse platelets upon illumination with an appropriate laser light source (see FIG. 40). Cells (1×10⁷/g of BWT) will be subsequently injected intravenously into mice bearing WT mouse (control) or the “humanized” A1 domains and their behavior visualized in the microcirculation using an intravital microscope (Zeiss, Axiotech Vario; IV500, Mikron Instruments, San Diego, Calif.; and the like) equipped with an iXON EM camera or a silicon-intensified camera (VE1000SIT; Dage mti, Michigan City, Ind.), a Yokogawa CSU22confocal head, and a 488 nm laser line (Andor Technology, Revolution series). A Xenon arc stroboscope (Chadwick Helmuth, El Monte, Calif.) will serve as the light source and fluorescent cells will be viewed through 60× or 100× water immersion objectives (Acroplan, Carl Zeiss Inc.). A tethered platelet will be defined as a cell establishing initial contact with the vessel wall (FIG. 33A, panel 2-3; FIG. 33B). The translocating fraction will be defined as number of tethered platelets that move at a velocity significantly lower than the centerline velocity for >1 s. The sticking fraction will be defined as the number of translocating cells that become stationary for >30 s post-tethering. Second order arterioles (up to 50 μm in diameter) will be evaluated for platelet interactions before and after the injury. Evaluation of platelet circulation in larger arterioles may be less accurate secondary to hemoglobin-mediated quenching of fluorescence emitted from platelets traveling in an area of the blood stream distal to the focal plane of the objective. Epi-illumination will only be used during video recordings to minimize possible phototoxic effects on tissue.

A role for GPIb alpha as well as the collagen (α2β1) and the fibrinogen (αIIb β3) receptors can be evaluated by using function-blocking antibodies to these proteins. Moreover, FDA approved anti-thrombotics (such as clopidogrel and tirofiban) can be examined as to whether the drugs inhibit human platelets from forming a thrombus in vivo, validating the mouse model for use in pre-clinical screening. The effect that antibodies and drugs have on altering the interaction between GPIb alpha-VWF-A1 interaction is determined by evaluating whether thrombus formation in the proposed mice is reduced or augmented upon arteriolar injury (FIG. 34).

For all experiments, the centerline erythrocyte velocity (Vrbc) is measured using an optical doppler velocimeter (Microcirculation Research Institute, Texas A&M College of Medicine, College Station, Tex.) prior to and after inducing the injury. Shear rate (SR) is then calculated based on Poiseulle's law for a Newtonian fluid: SR=8(Vmean/Dv), where Dv is the diameter of the vessel and Vmean is estimated from the measured Vrbc (Vmean=Vrbc/1.6).

Characterization of thrombus formation: Thrombus formation can be characterized as follows: (1) Early individual platelet interactions with the damaged vessel wall (number of fluorescently labeled human platelets that attach during the first minute post-injury); (2) time required for thrombus generation of >20 μm diameter; (3) the ability of thrombi to remain at the initial site of vascular injury and not break free (measure of stability); (4) time until vessel occlusion; and (5) site of vessel occlusion, that is, at the site of injury or downstream from it. Platelet-vessel wall interactions can be viewed through 40× or 60× water immersion objectives. To standardize in vivo conditions, the velocity of flowing blood (shear rate) pre-injury is determined by measuring the centerline erythrocyte velocity (Vrbc) using an optical doppler velocimeter. Shear rate (SR) can then be calculated based on Poiseulle's law for a Newtonian fluid: SR=8×(Vmean/Dv), where Dv is the diameter of the vessel and Vmean is estimated from the measured Vrbc (Vmean=Vrbc/1.6). Vessel and thrombus diameters are measured using imaging software (ImagePro Plus).

Administration of antibodies: Function-blocking monoclonal antibodies 6D1 (anti-human GPIb alpha), 6F1 (anti-human α2β1) and 7E3 (anti-human αIIbβ3) have been generously provided by Dr. Barry Coller (Rockefeller University, NY). All antibodies are converted to F(ab′)2 fragments to limit Fc receptor interactions in vivo. An intravenous dose of 10 μg/g body weight is given approximately 10 minutes after the injection of human platelets but 30 minutes prior to inducing vascular injury. Non-function blocking antibodies to these receptors is used as negative controls and administered under identical conditions. To ensure optimal ligand availability for the collagen and fibrinogen receptors on human platelet, mice possessing the A1 domain mutation have been bred with animals genetically deficient in α2β1 or αIIbβ3. Thus, endogenous platelets in these animals not only have a reduced ability to interact with the VWF-A1 domain, but also are incapable of binding to collagen or fibrinogen, respectively. Although human platelets have been shown to circulate in mice for a maximum of 24 hours, we can ensure that an equivalent percentage of human platelets are present at the time of vascular injury under each experimental condition (Xu et al. J. Clin. Invest. 2006; 116: 769). Thus, 50 μl is obtained from an inserted venous catheter and flow cytometric analysis will be performed to determine the percentage of circulating fluorescently-labeled human platelets.

Administration of drugs: In comparison to aspirin, clopidogrel (Plavix) is the second most commonly used anti-thrombotic drug that targets one of the ADP receptors (P2Y12) on platelets, causing irreversible inhibition (Hankey et al. Med. J. Aust. 2003; 178:568). ADP is a potent mediator of platelet activation and aggregate formation, and thus considerable effort and funds have been devoted to inhibiting this activation pathway in platelets. Clopidogrel was approved by the FDA in 1997 for clinical use and was found to be of benefit in the secondary prevention of major vascular events in patients with a history of cerebrovascular and coronary artery diseases and major cardiac events post coronary artery stent placement (Gachet et al. Semin. Thromb. Hemost. 2005; 31:162). Disadvantages of this drug are: 1) It must be metabolized in the liver to generate an active metabolite, thus limiting its effectiveness in acute settings, and 2) irreversible inhibition that results in a marked prolongation of bleeding time.

Clopidogrel has been shown to reduce thrombus size and delay its formation in mice with a maximal effective dose of 50 mg/kg given the day before and 2 hours prior to experimentation (Lenain et al. J. Throm. Haemost. 2003; 1:1133). This drug will be obtained from the hospital pharmacy and tablets will be dissolved in sterile water for oral administration. Control animals will receive water in lieu of drug. The effectiveness of this treatment regime will be confirmed by first measuring the responsiveness of platelets isolated from drug-treated WT animals to ADP-induced aggregation using an optical aggregometer (Chrono-Log Corp.) as previously described (Leon et al. J. Clin. Invest. 1999; 104:1731). As our mutant VWF-A1 domain mice also have a defect in platelet aggregation, these animals cannot be used for the purpose of testing to ADP-induced aggregation ex-vivo. However, this additional phenotype will be advantageous for us as it limits potential competition between human and mouse platelets for binding to ligands exposed at sites of vascular injury. Human platelets will be administered 30 minutes prior to vascular injury and 50 μl of blood drawn to determine the percentage of circulating cells as described above. Platelet rich plasma will also be purified from control and drug treated animals that receive human platelets to evaluate the effectiveness of clopidogrel on preventing ADP-induced aggregation of these cells ex-vivo.

Tirofiban (Aggrastat) is a non-peptide inhibitor of the fibrinogen receptor αIIbβ3 that limits the ability of platelets to form aggregates, an event required for thrombus progression. It has a plasma half-life of approximately 2 hours but only remains bound to platelets for seconds, thus necessitating continuous intravenous administration. It is currently approved for short-term treatment of patients with acute coronary syndrome that require interventional catheterization. Thus, the animals will be dosed based on that given for interventional procedures such as angioplasty, which consists of a 25 μg/kg bolus over 3 minutes followed by a continuous maintenance infusion of 0.15 μg/kg/min until the completion of the experiment (Valgimigli et al. JAMA. 2005; 293:2109). Human platelets will be administered 30 minutes prior to vascular injury and 50 μl of blood drawn to determine the percentage of circulating cells as described above.

Platelet donors. Mice are used as platelet donors. A means to evaluate murine platelet interactions with wild type and mutant VWF-A1 proteins is via in vitro flow chamber assays. Blood from ˜10 mice are required to purify adequate numbers of platelets per assay. Blood from donor animals is obtained from the retro-orbital plexus using a heparinized glass pipette. Mice will be anesthetized with Ketamine and Xylazine prior to the procedure and are euthanized by CO₂ inhalation upon completion.

Bleeding time for human platelet induced hemostasis. This assay is carried out as described above.

Solution-phase binding assay. For type 2B mutant VWF, its capacity to bind to platelet GPIb alpha in solution can be determined. Plasma is harvested from these mice and VWF purified. Various concentrations of the plasma glycoprotein will be indirectly labeled using a non-function blocking, ¹²⁵I-labeled mAb to its A1 domain as previously described (67). After a 30 min. incubation, a quantity of this mixture will be incubated with platelets purified from beta 3 deficient mice so to prevent integrin-mediated binding to VWF. After incubation period of 1 hour, an aliquot of this mixture will be added to a sucrose gradient and centrifuged to pellet the platelets. Radioactivity associated with the pellet vs. supernatant will be determined in a γ-scintillation counter, and the binding estimated as the percent of total radioactivity.

Example 4 Defining the In Vivo Role of the von Willebrand Factor A1 Domain by Modifying a Species-Divergent Bond

Proteins containing von Willebrand Factor (VWF) A domains contribute to human health and disease by promoting adhesive interactions between cells (Whittaker, C. A., & Hynes, R O. Mol. Biol. Cell. 13, 3369-3387 (2002)). The VWF-A1 domain is thought to play a critical role in hemostasis by initiating the rapid deposition of platelets at sites of vascular damage by binding to the platelet receptor glycoprotein Ib alpha (GPIbα) at high shear rates (Roth, G. J. Blood 77, 5-19 (1991); Cruz, M. A., et al., J. Biol. Chem. 268, 21238-21245 (1993); Sugimoto, M. et al., Biochemistry 30, 5202-5209 (1991); Pietu, G. et al., Biochem. Biophys. Res. Commun. 164, 1339-1347 (1989)). Although congenital absence of VWF in humans has established a role for this plasma glycoprotein in hemostasis (Sadler, J. E. et al. J. Thromb. Haemost. 4, 2103-2114 (2006)), the contribution of its A1 domain in clot formation has been questioned in a mouse model of vascular injury (Denis, C. et al. Proc. Natl. Acad. Sci. USA 95, 9524-9529 (1998)).

In this example, murine plasma VWF or its A1 domain fails to support significant interactions with human platelets (and likewise human VWF with murine platelets) under flow conditions. Atomic models of GPIbα-VWF-A1 complexes suggest that the structural basis for this behavior arises primarily from an electrostatic “hot-spot” at the binding interface. Introduction of a single point mutation within this region of murine VWF-A1 is sufficient to switch its binding specificity from murine to human platelets. In addition, introduction of a single point mutation within the electrostatic “hot-spot” region of human VWF-A1 is sufficient to switch its binding specificity from human to murine platelets. Moreover, mice possessing the 1326R>H mutation in their VWF have a bleeding phenotype distinct from VWF-deficient animals, and can be corrected by the administration of human platelets. Mechanistically, mutant animals can generate but not maintain thrombi at sites of vascular injury, whereas those infused with human platelets can form stable thrombi, a process that relies on GPIbα-VWF-A1 interaction. Thus, interspecies differences at protein interfaces can provide insight into the biological importance of a receptor-ligand bond, and aid in the development of an animal model to study human platelet behavior and drug therapies.

Methods

Generation of VWF^(1326R>H) mice. The VWF^(1326R>H) targeting vector (FIG. 38A) was prepared from a 129/SvJ mouse genomic library. The clone was identified by PCR using primers specific for exon 28 of the mouse VWF gene and sequence fidelity of the region to be targeted validated by comparison to published sequence for chromosome 6 (GenBank accession number NW_(—)001030811). The targeting vector is identical to the corresponding region in the mouse genome, except the 1326R>H mutation was created in exon 28 and the Neo cassette flanked by loxP sites was inserted into intron 28. This resulted in the loss of an EcoRV site and the introduction of a new EcoR1 and two new XhoI sites. The construct was electroporated into an embryonic stem (ES) cell line, and potential clones identified by continued growth of cells in G418 and Gancyclovir supplemented media. DNA was isolated from surviving colonies, digested with EcoRI, and screened by Southern analysis using a 1.5 kb probe (A) corresponding to a DNA sequence downstream of the targeting construct. Chimeric mice generated from VWF^(1326R>H) targeted ES cell lines were subsequently bred to a Cre transgenic mouse (C57BL/6 background) and animals containing the 1326R>H mutation, but without the Neo cassette, subsequently identified by both PCR and Southern analysis. WT and homozygous animals were the product of matings between heterozygous mice.

Analysis of VWF transcripts, antigen levels, multimers, and collagen binding. Detection of transcripts from the A1-A2-A3 domains of murine VWF was performed by RT-PCR. Briefly, mRNA was isolated from lung tissue harvested from either homozygous VWF-A1^(1326R>H) mice or aged-mated WT littermate controls (Oligotex®, Qiagen). Generation of cDNA and PCR-amplification of desired transcripts was performed using SuperScript™ One-Step RT-PCR (Invitrogen) and oligos specific for the A domains of VWF.

Functional factor VIII levels were determined by a mechanical clot detection method using the STA automated coagulation analyzer (Diagnostica Stago, Parsippany, N.J.). A log-log calibration curve was established by measuring the activated partial Thromboplastin time (aPTT) of varying dilutions of reference plasma. The aPTT of a 1:10 dilution of sample plasma mixed with factor VIII deficient plasma was determined, compared to the calibration curve, and the activity expressed as a percent of normal.

Evaluation of VWF antigen levels was performed as previously described (Denis, C. et al. Proc. Natl. Acad. Sci. USA 95, 9524-9529 (1998)). For multimer analysis, plasma from sodium citrate treated whole blood was diluted 1:5 in electrophoresis sample buffer (final concentration 10 mM Tris-HCl pH 8.0, 2% SDS, 1 mM EDTA) and heated at 56° C. for 30 minutes. Electrophoresis was carried out overnight (64 volts, 15° C.) through a horizontal SDS-agarose gel in 1.2% agarose (Ruggeri, Z. M. & Zimmerman, T. S. Blood 57, 1140-1143 (1981)). The gel was then electrophoretically transferred (150 mA, 90 minutes) to Immobilon (Millipore) followed by blocking (2 h) with 5% powdered milk in TBST (Tris HCl pH 8.0, 0.15M NaCl, 0.05% Tween-20). The membrane was incubated with a 1:500 dilution of rabbit anti-human VWF antiserum (DAKO) for 1 h, washed in TBST, and then incubated with a 1:10,000 dilution of HRP-conjugated mouse anti-rabbit IgG (Calbiochem). Bands were subsequently detected by chemiluminescence system (GE Healthcare). For comparison, a sample containing pooled human plasma from normals or patients with type 2B VWD was also loaded on the gel. Binding of VWF to surface-immobilized collagen was performed as previously described (Smith, C. et al. (2000) J. Biol. Chem. 275, 4205-4209). Briefly, 100 μg/ml of acid soluble type I collagen from human placenta (Sigma) was added to a 96 well microtiter plate and allowed to incubate overnight (4° C.). After washing and blocking with TBS containing 3% BSA and 0.05% Tween 20, varying concentration of platelet poor plasma harvested and pooled from WT, homozygous VWF^(1326R>H), and VWF deficient mice was added to the wells and incubated for 1 h (37° C.). Wells were then washed and bound VWF detected by an ELISA as described above.

Ex vivo platelet adhesion studies. Experiments were performed in a parallel-plate flow chamber as previously described (Offermanns, S. (2006) Circ. Res. 99, 1293-1304). For studies involving plasma VWF, a polyclonal anti-VWF antibody (Dako) was absorbed overnight (4° C.) to a six well tissue culture plate. Subsequently, the plate was washed and non-specific interactions blocked by the addition of TBS containing 3% BSA, pH 7.4 (1 h, 37° C.). Human or murine plasma obtained from heparinized whole blood was added and the plates placed at 37° C. for an additional 2 h. Generation, purification, and surface-immobilization of recombinant VWF-A1 proteins was performed as previously described (Doggett, T. A. et al. Biophys. J. 83, 194-205 (2002)). Both human and murine VWF-A1 constructs consist of amino acid residues 1238 to 1471, with a single intra-disulfide bond formed between residues 1272 and 1458 and were generated in bacteria. Citrated whole blood (150 μl) collected via cardiac puncture from anesthetized homozygous VWF^(1326R>H) or WT mice or from venopuncture from human volunteers was perfused over the immobilized substrates at a wall shear rate of 1600 s⁻¹ for 4 min, followed by washing with Tyrode's buffer under the identical flow conditions. The number of platelets attached per unit area (0.07 mm²) and translocation velocities were determined by off-line analysis (Image-Pro Plus, Media Cybernetics). For GPIbα inhibition studies, the function-blocking mAb 6D1 (20 μg/ml) or mAb SZ2 (20 μg/ml; Beckman Coulter) was added to anticoagulated human blood for 30 min prior to use. Experiments were performed in triplicate on two separate days. An ELISA was used to ensure equivalent coating concentration of plasma and recombinant proteins (Denis, C. et al. (1998) Proc. Natl. Acad. Sci. USA 95, 9524-9529).

In vivo thrombus formation. Administration of anesthesia, insertion of venous and arterial catheters, fluorescent labeling and administration of human platelets (5×10⁸/ml), and surgical preparation of the cremaster muscle in mice have been previously described (Doggett, T. A. et al. Biophys. J. 83, 194-205 (2002); Diacovo, T. G., et al., Science 273, 252-255 (1996)). Injury to the vessel wall of arterioles (˜40-65 μm diameter) was performed using a pulsed nitrogen dye laser (440 nm, Photonic Instruments) applied through a 20× water-immersion Olympus objective (LUMPlanFl, 0.5 NA) of a Zeiss Axiotech vario microscope. Mouse platelet- and human platelet-vessel wall interactions were visualized using either bright field or fluorescence microscopy. The latter utilized a fluorescent microscope system equipped with a Yokogawa CSU-22 spinning disk confocal scanner and 488 nm laser line (Revolution XD, Andor™ Technology). The extent of thrombus formation was assessed for 2 min post injury and the area (μm²) of coverage determined (Image IQ, Andor™ Technology). For GPIbα or αIIbβ3 inhibition studies, the function-blocking mAb 6D1 or 7E3 (20 μg/ml), respectively (from B. Coller, Rockefeller University), was added to purified human platelets for 30 min prior to administration.

Tail bleeding assay. Bleeding times were measured in 7-week old mice after amputating 1 cm of the tail tip as previously described (Denis, C. et al. (1998) Proc. Natl. Acad. Sci. USA 95, 9524-9529). In studies involving human platelets, platelet concentrates were obtained from Columbia Presbyterian Hospital Blood Bank, washed and resuspended in normal saline (1.5×10⁹/300 μl) before administering through a catheter inserted into the right internal jugular vein. Tail cuts were performed 5 min after completion of the infusion of platelets. PLAVIX and ReoPro® were obtained from the research pharmacy at CUMC. For studies involving PLAVIX, animals received a 50 mg/kg oral dose of the drug the day before and 2 h prior to the administration of human platelets. ReoPro® was given initially as an intravenous bolus (0.25 mg/kg) 5 min after the administration of human platelets, followed by a continuous infusion (0.125 μg/kg/min) as per the manufacturer's recommendations.

Structural Modeling. There are three crystal structures of the GPIbα-VWF-A1 complex: two are WT except for mutated N-glycosylation sites in GPIbα (Fukuda, K. et al., (2005) Nat. Struct. Mol. Biol. 12, 152-159; Dumas, J. J. et al. (2004) J. Biol. Chem. 279, 23327-23334), and one is a gain-of-function mutant (Huizinga, E. G. et al. (2002) Science 297, 1176-1179). The structures have only small differences that are not the result of the presence of mutations or botrocetin binding (Fukuda, K., et al., (2005) Nat. Struct. Mol. Biol. 12, 152-159). Both N-glycosylation sites in human GPIbα lie on the well-ordered upper ridge of the LRR, 18 Å and 27 Å (Cα-Cα) from the nearest VWF-A1 residue, so their absence is unlikely to affect the structure of the complex. Murine GPIbα has no predicted N-glycosylation sites.

Human GPIbα contains sulfated tyrosines implicated in binding VWF within an acidic loop just C-terminal to the sequence included in the crystal structures. Murine GPIbα has a predicted sulfation site in the same loop, so that the differential binding of human vs. murine GPIbα to VWF-A1 is also likely to be small. The interfacial regions are otherwise highly conserved between species, with the exception of three salt bridges (See FIGS. 37C-G). The conformation of the β-switch region is highly constrained, as noted in the main text. The only change to a buried interfacial residue is M239T (human to mouse), which lies in an invariant pocket. Notably, the crystal structure of the human “gain-of-function” mutant, M239V, shows no perturbations in this region, and given that valine is isosteric with threonine, this species difference is unlikely to affect either the complex structure or interspecies binding.

We used a consensus model of the human complex to build the murine model. We first overlaid murine A1 onto human A1 by fitting the central β-sheets (RMSD 0.3 Å; within experimental error); the only notable difference is the location of helix α4, which is shifted by 2-3 Å away from the GPIbα interface in the mouse owing to a larger residue on the buried face of this helix. For the GPIbα model, only the side-chains were altered, since the human and murine LRRs have identical lengths. Consensus rotamers with minimal steric clashes were chosen, followed by manual adjustment where necessary to create sensible van der waals interactions and H-bonding, using TURBOFRODO (Bio-Graphics, Marseille, France). Molecular overlays were optimized using LSQKAB (Collaborative Computational Project, No. 4. Acta Crystallogr. D50, 760-763 (1994)); molecular figures were created using MOLSCRIPT (Esnouf, R. M. J. Mol. Graph. Model. 15, 132-136 (1997)) and OPENGL (http://www.rush3d.com/reference/opengl-bluebook-1.0)

Statistics. An unpaired, two-tailed Student t test was used for multiple comparisons.

Results and Discussion

As the interaction between GPIbα and VWF-A1 is a prerequisite for effective thrombus formation in the arterial circulation, we first tested the ex vivo ability of surface-bound murine plasma VWF or its recombinant A1 domain (rVWF-A1) to support human platelet adhesion under physiologically relevant flow conditions: that is at a shear rate exceeding 1000 s⁻¹ using a parallel-plate flow chamber (Ruggeri, Z. M. et al., (2006) Blood. 108, 1903-1910). The adhesive properties of VWF are tightly regulated such that it preferentially binds to platelets only when immobilized to sites of vascular injury and under hydrodynamic conditions encountered on the arterial side of the circulation (Sakariassen et al., (1979) Nature 279, 636-638; Ruggeri, Z. M. et al., (2006) Blood. 108, 1903-1910). Perfusion of human whole blood over murine plasma VWF or rVWF-A1 resulted in limited platelet deposition (10 to 25%, respectively) as compared with same-species controls (FIG. 36A-B). Similarly, human VWF proteins had a diminished capacity to support murine platelet accumulation under identical conditions (FIGS. 36C-D). This interspecies incompatibility would seem to preclude the study of human platelet behavior in a mouse model of arterial thrombosis.

In order to gain insight into the structural origins of this species incompatibility, we built models of murine-murine and human-murine GPIbα-VWF-A1 complexes based on the crystal structures of the human complex (Fukuda, K., et al., Nat. Struct. Mol. Biol. 12, 152-159 (2005); Dumas, J. J. et al., J. Biol. Chem. 279, 23327-23334 (2004); Huizinga, E. G. et al., Science 297, 1176-1179 (2002)) and human and murine VWF-A1 (Fukuda, K., et al., Nat. Struct. Mol. Biol. 12, 152-159 (2005)) (FIG. 37A-E; see Methods).

The A1 domain comprises a Rossmann-like fold with a central, mostly parallel β-sheet flanked on both sides by α-helices (Fukuda, K., et al., Nat. Struct. Mol. Biol. 12, 152-159 (2005)). Human and murine VWF-A1 share considerable sequence (86% identity) and structure homology; in fact, the β-sheets of both species are identical within experimental error (a root mean square difference of 0.33 Å for Cα-atoms). The only major difference is the location of helix α4 (nomenclature as previously described in Dumas, J. J. et al., J. Biol. Chem. 279, 23327-23334 (2004)), which is shifted 2-3 Å away from the GPIbα binding site in the mouse, owing to a difference in a buried hydrophobic residue (FIG. 37 A-B). Although neither the structure of murine GPIbα nor its complex with the VWF-A1 domain are known, the high sequence similarity of the murine and human proteins (including the complex interface), as well as the rigid architecture of the leucine-rich repeats (LRR) of GPIbα, provide high confidence that their 3D structures will be highly homologous.

In the complexes, the major contact region involves the “β-switch” region (residues 227 to 241 in the C-terminal flank of GPIb alpha), which forms a β-hairpin that augments the β-sheet of the VWF-A1 domain. On its other side, this region of GPIbα packs tightly against the concave face of the LRR, which highly constrains it movement. Residues in mouse and human are mostly invariant on both sides of this interface. Notable exceptions are at position 1326 in VWF-A1, which is histidine (H) in humans versus an arginine (R) in mouse, and at position 238 in GPIb alpha, which is alanine (A) in humans versus an aspartic acid (D) in mouse (FIGS. 37C and 37D). A model of the murine complex suggests that these changes are complementary, since D238 can form an intermolecular salt-bridge with R1326; D238 in murine GPIb alpha also shields the positively charged flanking lysine (K) at position 231 (a conserved residue in both species) from unfavorable interactions with R1326 in murine VWF-A1. This salt-bridge cannot form in the human complex due to the presence of a histidine at 1326. However, an intermolecular salt-bridge can occur between R1395 and E225 located at the top of the human complex, which may compensate for this loss (FIG. 37D). No such interaction can occur in the murine complex (FIG. 37C).

In the human GPIb alpha-murine VWF-A1 interspecies complex, we predict that the two positively charged residues (GPIb alpha K231 and VWF-A1 R1326) create an electrostatic clash that impedes binding, owing to the lack of a negatively charged group at position 238 (FIG. 37E). In the murine GPIb alpha-human VWF-A1 interspecies complex, however, no such electrostatic clash occurs despite the absence of the salt-bridge. There is, however, an overall change in net charge in the binding interface compared with the murine GPIb alpha-murine VWF-A1 complex (FIG. 37F). This, together with the loss of critical salt-bridges, most likely accounts for the reduced interaction between mouse platelets and human VWF (See Tables 5 and 6).

TABLE 5 Predicted effect of species differences in residues on the human GPIb alpha-murine VWF-A1 interspecies complex. hGPIb-alpha hGPIb alpha- mVWF-A1 partner mVWF-A1 Reason R1326 A238 (−) Permits electrostatic clash of R1326 with K231 in GPIb alpha E1330 K237 (+) New salt-bridge E1330-K237 G1370 none 0 No interactions R1395 E225 (−) Loss of salt-bridge (shifts position) (+) = net positive, (−) = net negative, 0 = minimal effect compared with syngeneic complexes.

TABLE 6 Predicted effect of species differences in residues on the murine GPIb alpha-human VWF-A1 interspecies complex. hGPIb- mGPIb-alpha alpha- hVWF-A1 partner mVWF-A1 Reason H1326 D238 (−) Loss of R1326-D238 salt- bridge G1330 K237 (−) Loss of E1330-K237 salt- bridge S1370 none 0 No interactions R1395 N225 (+) New polar interactions with (shifts position) R1395 (+) = net positive, (−) = net negative, 0 = minimal effect compared with syngeneic complexes.

To explore the importance of the electrostatic mismatches in destabilizing the interspecies complexes, we substituted human residues into murine rVWF-A1 at positions 1326 (R>H), 1330 (E>G), and 1370 (S>G), and analyzed the ability of the mutant proteins to support human platelet accumulation under flow. As expected, amino acid substitutions at positions 1330 (predicted to remove a salt-bridge) and 1370 (predicted to have no effect) failed to promote the interaction between murine rVWF-A1 and human GPIbα. However, the 1326R>H mutation, which eliminates the electrostatic clash with K231, rendered murine A1 capable of supporting interactions at a level comparable to its wild-type (WT) human counterpart (FIG. 37G). Similarly, conversion of 1326H>R in the human rVWF-A1 protein promoted the binding of mouse platelets, while the reverse substitution in its murine counterpart reduced adhesion by 75%. That a single residue change is sufficient for shifting the binding preferences across species supports the notion that this contact region is a “hot-spot” in the protein interface (Bogan, A. A. & Thorn, K. S. J. Mol. Biol. 280, 1-9 (1998)).

In order to determine the ability of full-length murine VWF containing the 1326R>H mutation (VWF^(1326R>H)) to support human platelet interactions and ultimately thrombus formation in vivo, we genetically modified mice to express VWF^(1326R>H) (FIG. 38B-C). Both homozygous and heterozygous animals were viable, fertile, born at the expected Mendelian ratio, and had platelet counts comparable to WT littermate controls. Moreover, reverse transcription-PCR(RT-PCR) of lung tissue from mutant mice with primers specific for the A1, A2, and/or A3 domains of VWF amplified cDNAs of the correct size and of similar intensity as compared to WT littermate controls (FIG. 39A). VWF antigen levels, Factor VIII function, as well as VWF multimer pattern in homozygous mutant plasma were found to be equivalent to WT controls (FIG. 39B-C). These results indicate that VWF gene translation, transcription, and posttranslational modifications were not perturbed by our targeting strategy. The ability of plasma VWF to bind to collagen was also not affected by the introduction of the point mutation.

Hemostasis relies on platelet adhesion and activation at sites of vascular injury, which ultimately results in the formation of a hemostatic plug. To demonstrate the importance of VWF-A1 in this process, we measured bleeding times for mice possessing the 1326R>H mutation by removing 1 cm of distal tail (FIG. 41A). In contrast to their WT counterparts, the vast majority of homozygous VWF^(1326R>H) mice were incapable of forming an effective hemostatic plug, as they continued to bleed profusely throughout the duration of the experiment (10 min). Moreover, a smaller but statistically significant increase in bleeding time was noted for heterozygous animals (1.9-fold compared with WT; P=0.0055). Thus, disruption of a single salt bridge between murine VWF-A1 and GPIbα is sufficient to impair hemostasis.

To gain insight into how the 1326R>H mutation alterations hemostasis, we evaluated murine platelet adhesion at sites of vascular damage in vivo. A laser-induced vascular injury model was utilized to initiate platelet deposition in arterioles located in the microcirculation of the cremaster muscle of WT and homozygous mutant mice (Furie, B. & Furie., B. C. (2005) J. Clin. Invest. 115, 3355-3362). Although VWF^(1326R>H) animals can initially form thrombi that fill the vessel lumen, they rapidly dissipated under the prevailing hydrodynamic conditions (FIG. 40). By contrast, thrombi in WT mice continue to enlarge and eventually occlude blood flow under identical conditions.

To demonstrate that the removal of the electrostatic clash between residues 1326 and 231 in murine VWF-A1 and human GPIb alpha, respectively, promotes substantial interactions between this chimeric receptor-ligand pair, we perfused human whole blood over surface-immobilized plasma VWF obtained from mice homozygous for VWF^(1326R>H) Remarkably, mutant murine VWF bound human platelets at levels comparable with its human counterpart (FIG. 41B). Moreover, a function-blocking antibody 6D1, which binds exclusively to human GPIbα at the fourth LRR (Shen, Y. et al. (2000) Blood. 95, 903-910), inhibited platelet adhesion, demonstrating a key role for the platelet receptor in adhesion to VWF^(1326R>H).

By contrast, the antibody SZ2 that recognizes the anionic-sulfated tyrosine sequence of GPIbα (residues 276 to 282) had a minimal affect on platelet accumulation, results consistent with a previous report (Fredrickson et al., (1998) Blood. 92, 3684-3693). The A1 domain also possesses the ability to support the movement of attached platelets in the direction of the prevailing hydrodynamic force owing to rapid and reversible interactions with GPIbα (Savage et al., (1996) Cell 84, 289-297; Doggett, T. A. et al. (2002) Biophys. J. 83, 194-205). We therefore compared translocation velocities of human platelets on either human or mutant murine VWF. Translocation velocities of human platelets on either homozygous mutant murine or native human plasma VWF were also similar (3.5±0.1 μm/sec vs. 3.2±0.1 μm/sec, respectively; mean±s.e.m, n=4), demonstrating that VWF^(1326R->H) functions in a manner indistinguishable from its human counterpart.

We next tested the ability of murine VWF^(1326R>H) to support human platelet adhesion in vivo. The ability of human platelets to preferentially support thrombus formation was monitored simultaneously by labeling purified human cells with BCECF ex-vivo, and mouse platelets with rhodamine 6G by intravenous administration. Fluorescently-labeled human platelets were infused continuously via a catheter inserted into the femoral artery, resulting in a high local concentration of these cells within the microcirculation of the cremaster muscle. Their behavior in response to laser-induced vascular injury was monitored in real-time using confocal intravital microscopy (Furie, B. & Furie., B. C. (2005) J. Clin. Invest. 115, 3355-3362). Upon induction of laser damage to the vessel wall of arterioles in mice homozygous for VWF^(1326R>H) human platelets rapidly adhered to the site of injury, forming large thrombi composed mainly of human cells (91.7±1.2%; mean±s.e.m) (FIG. 41C-D); average thrombus size was 8,950±1,620 μm² (mean±s.e.m.). In WT mice, by contrast, human platelets had only a limited capacity to bind to the damaged vessel wall, accounting for only 5.7±0.4% of total thrombus area (mean±s.e.m.) (FIG. 41C-D).

Consistent with the critical role of platelet GPIb alpha in mediating interactions with VWF-A1, pre-treatment of human platelets with mAb 6D1 greatly reduced thrombus size in the vasculature of VWF^(1326R>H) mice (265±125 μm²; mean±s.e.m.) (FIG. 41E). This was also validated by our observations that human platelet deposition at sites of arterial injury is limited in VWF-deficient mice (185±35 μm²; mean±s.e.m.), demonstrating that the A1 domain of this plasma protein serves as the major ligand for GPIbα in our humanized animal model of thrombosis.

Although GPIbα initiates platelet deposition at arterial shear rates, it is ultimately the platelet integrin αIIbβ3 that supports thrombus growth by promoting platelet-platelet interactions. The contribution of human αIIbβ3 in this process is demonstrated by the ability of the function blocking antibody 7E3 to also limit thrombus size (529±150 μm²) (FIG. 41E). Confirmation that the interaction formed between human platelets and murine VWF^(1326R>H) is sufficient to promote effective hemostasis is provided by the ability of infused human cells to restore bleeding times in mutant mice to a level observed for their WT counterparts (182±14.5 s vs. 131.5±11.2 s, respectively; mean±s.e.m.) (FIG. 41F). Importantly, human platelet-induced hemostatic clot formation can be completely disrupted in these animals by the preadministration of either clopidogrel (PLAVIX), an inhibitor of ADP-induced platelet activation, or abciximab (ReoPro®), a Fab fragment of the chimeric human-mouse monoclonal antibody 7E3, which blocks the function of αIIbβ3 on human but not murine platelets (Hankey, G. J. & Eikelboom, J. W. Antiplatelet drugs. Med. J. Aust. 178, 568-574; Bennett, J. S. (2001) Annu. Rev. Med. 52, 161-184) (FIG. 41F).

In summary, these studies demonstrate how one can effectively utilize atomic models of interspecies complexes to identify a binding hot spot where a disproportionate amount of the binding free energy is localized, such that a single amino acid substitution significantly affects the interaction (Bogan, A. A. & Thorn, K. S. (1998) J. Mol. Biol. 280, 1-9), and in this case switches species specificity. Moreover, a subtle and localized change of this nature limits the possibility of inducing structural perturbations that impact on the function of other domains contained within VWF. These show that human platelet adhesion to VWF^(1326R>H) is dependent on GPIbα binding to VWF-A1, with other potential ligands for this receptor playing a subservient role in this process (Bergmeier, W. et al. (2006) Proc. Natl. Acad. Sci. USA. 103, 16900-16905). The reliance of human thrombus formation on the integrin αIIbβ3, as well as the ability of the FDA approved drugs PLAVIX and ReoPro® to impair human platelet-mediated hemostasis indicate that downstream adhesive and activation events known to be critical for clot formation and stability are intact in the mutant VWF animals. Thus, we anticipate that the VWF^(1326R>H) knock-in mice will prove useful in the preclinical evaluation of new antithrombotic therapeutics designed ultimately for human use. These results also have implications for advancing both knowledge of human platelet biology and in preclinical testing of antithrombotic therapies in vivo.

Example 5 Use of “Humanized” VWF-A1 Animal for Developing Technologies to Image Sites of Occult Bleeding or Thrombus Formation in Humans

Perfluorocarbon Nanoparticle based imaging platform. The ability of a VWF-A1 mutant animal, such as our 1326R>H mutant mouse, to generate thrombi composed of human platelets at sites of vascular injury in vivo, provides a means for developing imaging technologies designed to detect sites of occult bleeding or thrombus formation in humans. For example, such technologies may prove useful expediting the discovery of sites of internal bleeding in humans as a result of injuries obtained form a motor vehicle accident. Similarly, it may be useful in detecting injuries obtained in a military battle. Suitable probes include antibodies, small molecules, peptides that recognize molecules expressed on human platelets or the various domains of VWF. However, coupling contrast agents directly to antibodies is cumbersome and insufficient for detection of such complexes in the body by various imaging modalities (i.e. MRI) due to low signal to noise output. Thus, an ideal candidate for detection would not only preserve the specificity associated with monoclonal antibodies, small molecules, or peptides but also have the following properties: 1) high signal-to-noise ratio, 2) long circulating half-life, 3) acceptable toxicity profile, 4) ease of use and production, and 5) compatibility with standard commercially available imaging modalities. Perfluorocarbon Nanoparticle (PNP) may provide the answer. This proposal will take advantage of a novel nanoparticle contrast agent that can be imaged by ultrasound, magnetic resonance, and nuclear imaging (Lanza et al., (2000) Invest Radiol, 35: 227-234; Lanza et al., (1997) Ultrasound Med Biol, 23: 863-870; Yu et al., (2000) Magn Reson Med. 44(6):867-72). This agent is a small (˜150-250 nanometer diameter), lipid encapsulated, perfluorocarbon emulsion that can be administered by vein. Importantly, monoclonal antibodies as well as small molecules and peptides that recognize platelets and/or VWF can be covalently coupled to PNPs. Moreover, PNPs can also be potentially used for targeted drug delivery (FIG. 42).

PNPs have been shown to remain stable in the circulation with a half-life of >1 hour, which permits rapid binding and local contrast enhancement sufficient for diagnostic imaging within 30-60 minutes. PNPs are cleared by the liver and spleen, and are similar to “artificial blood” formulations used to enhance oxygen, which have acceptable safety profiles for clinical use at 10 times greater dose than would be required for targeted contrast enhancement. In addition, perfluorocarbon to be used in this study (perfluorooctylbromide) has an extensive tract record for human safety in clinical trials (i.e. Oxygent, Alliance Pharmaceuticals). Thus, this nanoparticle platform provides an ideal opportunity to prove that contrast agents can be targeted specifically to sites of human thrombus formation.

Preparation of Fluorescently-Labeled Antibody Targeted Nanoparticles.

The basic method for formulating perfluorocarbon nanoparticles comprised of perfluorooctyl bromide (40% w/v), a surfactant co-mixture (2.0%, w/v) and glycerin 9(1.7%, w/v) has been well described (Lanza et al., (2000) Invest Radiol, 35: 227-234; Lanza et al., (1997) Ultrasound Med Biol, 23: 863-870).

Briefly, the surfactant co-mixture is dissolved in chloroform/methanol, evaporated under reduced pressure, dried in a 50° C. vacuum oven, and finally dispersed into water by sonication. The suspension is combined with perfluorocarbon and then emulsified at 20,000 PSI. Fluorescent nanoparticles are manufactured by including in the lipid mixture 0.1 mole % Fluorescence-FITC or PE prior to the emulsification step. Coupling of monoclonal antibodies involves the introduction of a sulflhydrl group onto the protein by modification of amines with N-succinimidyl S-acetylthioacetate (SATA), which then is reacted with nanoparticles containing activated maleimide. We coupled an antibody that recognizes the human, but not mouse, platelet receptor alphaIIb beta₃ and determined the ability of FITC-labeled PNPs to detect a thrombus composed of human platelets at a site of laser-induced vascular injury in the cremaster muscle of a mouse homozygous for the 1326R>H mutation. These antibody-coupled PNPs rapidly and selectively accumulated at the site of the developing human thrombus (FIG. 43).

Example 6 Identification of Small Molecules that Mitigate Binding Between GPIb Alpha and the VWF-A1 Domain

Small molecules, often with molecular weights of 500 or below, have proven to be extremely important to researchers to explore function at the molecular, cellular, and in vivo level. Such compounds have also been proven to be valuable as drugs to treat diseases, and most medicines marketed today are from this class (i.e. Aggrastat—see above). As the interaction between GPIb alpha and VWF-A1 is essential for the platelet deposition in damaged arterioles, it is a reasonable to assume that disruption of this adhesive event will inhibit or ameliorate thrombus formation. Moreover, we speculate that only partial inhibition is required to achieve this goal based on the phenotype of our mutant A1 domain mice, the inability to form stable thrombi in vivo.

Computational design based on the structure of the binary complex. Traditional approaches to small molecule discovery typically rely on a step-wise synthesis and screening program for large numbers of compounds to optimize activity profiles. Over the past decade, scientists have used computer models to aid in the development of new chemical agonists or antagonists as well as to better define activity profiles and binding affinities of such compounds. In particular, these tools are being successfully used, in conjunction with traditional research techniques, to examine the structural properties of existing compounds in order to predict their ability to alter the function of biologically relevant proteins. For this approach to be successful, one must have high quality crystal structures of the biological molecule(s) in order to generate an accurate 3-dimensional model so that it can then be used to identify binding regions for small molecules.

The structure of the binary complex formed when GPIb alpha binds to the A1 domain of VWF can be determined using such methods. For example, a mechanism by which the snake venom protein botrocetin enhances the interaction between GPIb alpha and the VWF-A1 in order to promote spontaneous platelet aggregation, resulting in death has been elucidated. Botrocetin was known to bind with high affinity to the A1 domain [see Table 7 for the crystallization data summary and Table 8 for atomic coordinate data], but was not thought to interact directly with GPIb alpha. This snake venom has the capacity to form a small, but distinct interface with this platelet receptor so to prevent its release from the A1 domain, thus facilitating platelet aggregation (FIG. 44). In a sense, nature has created a molecule that modifies the behavior of a known biological interaction, suggesting that one may be able to target man-made structures to this domain as well.

TABLE 7 Summary of Crystallization data (from PDB access no. 1AUQ and J Biol Chem (1998) 273 (17): 10396-10401) Structure Free enzyme (see Table 8) Space Group P6₁ Maximum resolution (Å) 2.3 Resolution range for refinement 10-2.3 Number of reflections 11,849 Completeness (%) 85.4 R factor² (%) 18.6 Free R factor² (%) 23.8 rms deviation in bond lengths (Å) 0.011 rms deviation in bond angles (°) 1.43

TABLE 8 Atomic Coordinates for Residues of a Crystal of murine VWF-A1 (SEQ ID NO: 10). CRYST1 86.395 86.395 68.125 90.00 90.00  120.00  P 61 6 ATOM 1 N ASP 498 22.142 52.453 −14.520 1.00 83.59 N ATOM 2 CA ASP 498 20.770 52.768 −14.026 1.00 83.86 C ATOM 3 C ASP 498 20.803 53.068 −12.522 1.00 81.16 C ATOM 4 O ASP 498 20.978 52.165 −11.696 1.00 81.93 O ATOM 5 CB ASP 498 19.821 51.597 −14.327 1.00 87.23 C ATOM 6 CG ASP 498 18.352 51.961 −14.133 1.00 88.94 C ATOM 7 OD1 ASP 498 18.022 53.171 −14.084 1.00 88.91 O ATOM 8 OD2 ASP 498 17.521 51.027 −14.033 1.00 90.11 O ATOM 9 N ILE 499 20.620 54.343 −12.190 1.00 76.43 N ATOM 10 CA ILE 499 20.649 54.829 −10.809 1.00 70.82 C ATOM 11 C ILE 499 19.237 55.126 −10.255 1.00 64.40 C ATOM 12 O ILE 499 18.888 56.265 −9.941 1.00 62.53 O ATOM 13 CB ILE 499 21.596 56.076 −10.724 1.00 75.17 C ATOM 14 CG1 ILE 499 21.872 56.476 −9.278 1.00 77.18 C ATOM 15 CG2 ILE 499 21.030 57.256 −11.527 1.00 77.54 C ATOM 16 CD1 ILE 499 23.010 57.485 −9.141 1.00 79.34 C ATOM 17 N SER 500 18.445 54.071 −10.111 1.00 58.81 N ATOM 18 CA SER 500 17.073 54.170 −9.616 1.00 52.67 C ATOM 19 C SER 500 16.771 52.914 −8.797 1.00 46.03 C ATOM 20 O SER 500 17.477 51.915 −8.928 1.00 46.10 O ATOM 21 CB SER 500 16.085 54.306 −10.779 1.00 55.03 C ATOM 22 OG SER 500 14.814 54.747 −10.320 1.00 55.43 O ATOM 23 N GLU 501 15.690 52.946 −8.017 1.00 39.21 N ATOM 24 CA GLU 501 15.341 51.839 −7.124 1.00 33.62 C ATOM 25 C GLU 501 13.826 51.632 −6.967 1.00 33.73 C ATOM 26 O GLU 501 13.055 52.575 −7.148 1.00 35.42 O ATOM 27 CB GLU 501 16.025 52.088 −5.772 1.00 27.09 C ATOM 28 CG GLU 501 15.928 53.517 −5.328 1.00 23.81 C ATOM 29 CD GLU 501 17.036 53.971 −4.394 1.00 22.26 C ATOM 30 OE1 GLU 501 18.150 53.442 −4.407 1.00 23.46 O ATOM 31 OE2 GLU 501 16.790 54.912 −3.645 1.00 19.78 O ATOM 32 N PRO 502 13.390 50.408 −6.585 1.00 32.77 N ATOM 33 CA PRO 502 11.980 50.038 −6.406 1.00 33.37 C ATOM 34 C PRO 502 11.257 50.886 −5.377 1.00 36.76 C ATOM 35 O PRO 502 11.881 51.657 −4.655 1.00 40.35 O ATOM 36 CB PRO 502 12.056 48.588 −5.911 1.00 32.84 C ATOM 37 CG PRO 502 13.426 48.154 −6.188 1.00 30.80 C ATOM 38 CD PRO 502 14.248 49.372 −5.994 1.00 33.53 C ATOM 39 N PRO 503 9.921 50.743 −5.283 1.00 38.50 N ATOM 40 CA PRO 503 9.095 51.495 −4.332 1.00 36.00 C ATOM 41 C PRO 503 9.217 50.861 −2.962 1.00 33.78 C ATOM 42 O PRO 503 9.351 49.642 −2.870 1.00 33.98 O ATOM 43 CB PRO 503 7.670 51.270 −4.844 1.00 35.94 C ATOM 44 CG PRO 503 7.844 50.708 −6.240 1.00 36.76 C ATOM 45 CD PRO 503 9.074 49.889 −6.137 1.00 37.21 C ATOM 46 N LEU 504 9.225 51.670 −1.910 1.00 32.48 N ATOM 47 CA LEU 504 9.284 51.120 −0.562 1.00 31.29 C ATOM 48 C LEU 504 7.869 50.821 −0.120 1.00 31.74 C ATOM 49 O LEU 504 7.663 50.104 0.847 1.00 34.42 O ATOM 50 CB LEU 504 9.857 52.133 0.427 1.00 27.31 C ATOM 51 CG LEU 504 11.346 52.216 0.633 1.00 20.47 C ATOM 52 CD1 LEU 504 11.608 53.354 1.577 1.00 16.31 C ATOM 53 CD2 LEU 504 11.865 50.886 1.213 1.00 22.70 C ATOM 54 N HIS 505 6.892 51.433 −0.779 1.00 32.33 N ATOM 55 CA HIS 505 5.508 51.234 −0.382 1.00 33.59 C ATOM 56 C HIS 505 4.574 50.872 −1.506 1.00 37.41 C ATOM 57 O HIS 505 4.980 50.817 −2.669 1.00 38.66 O ATOM 58 CB HIS 505 4.977 52.504 0.277 1.00 30.39 C ATOM 59 CG HIS 505 5.799 52.956 1.432 1.00 24.50 C ATOM 60 ND1 HIS 505 6.533 54.118 1.413 1.00 26.88 N ATOM 61 CD2 HIS 505 6.041 52.377 2.628 1.00 22.75 C ATOM 62 CE1 HIS 505 7.195 54.235 2.543 1.00 22.86 C ATOM 63 NE2 HIS 505 6.915 53.193 3.297 1.00 23.78 N ATOM 64 N ASP 506 3.323 50.612 −1.125 1.00 39.57 N ATOM 65 CA ASP 506 2.254 50.291 −2.072 1.00 41.22 C ATOM 66 C ASP 506 1.600 51.614 −2.482 1.00 39.47 C ATOM 67 O ASP 506 1.306 51.826 −3.651 1.00 42.48 O ATOM 68 CB ASP 506 1.231 49.325 −1.443 1.00 44.20 C ATOM 69 CG ASP 506 1.831 47.941 −1.106 1.00 47.68 C ATOM 70 OD1 ASP 506 2.837 47.508 −1.730 1.00 48.34 O ATOM 71 OD2 ASP 506 1.276 47.272 −0.209 1.00 49.92 O ATOM 72 N PHE 507 1.367 52.497 −1.507 1.00 38.50 N ATOM 73 CA PHE 507 0.800 53.830 −1.756 1.00 35.03 C ATOM 74 C PHE 507 2.016 54.527 −2.406 1.00 36.00 C ATOM 75 O PHE 507 3.086 54.637 −1.780 1.00 35.29 O ATOM 76 CB PHE 507 0.408 54.471 −0.418 1.00 32.75 C ATOM 77 CG PHE 507 −0.252 55.810 −0.552 1.00 32.71 C ATOM 78 CD1 PHE 507 −1.605 55.915 −0.866 1.00 33.60 C ATOM 79 CD2 PHE 507 0.471 56.969 −0.343 1.00 29.61 C ATOM 80 CE1 PHE 507 −2.216 57.157 −0.962 1.00 31.49 C ATOM 81 CE2 PHE 507 −0.132 58.205 −0.437 1.00 29.32 C ATOM 82 CZ PHE 507 −1.477 58.298 −0.750 1.00 33.05 C ATOM 83 N TYR 508 1.839 55.032 −3.624 1.00 35.32 N ATOM 84 CA TYR 508 2.955 55.578 −4.396 1.00 32.82 C ATOM 85 C TYR 508 2.445 56.499 −5.515 1.00 33.01 C ATOM 86 O TYR 508 1.484 56.154 −6.200 1.00 39.06 O ATOM 87 CB TYR 508 3.612 54.364 −5.053 1.00 27.12 C ATOM 88 CG TYR 508 5.027 54.464 −5.529 1.00 31.57 C ATOM 89 CD1 TYR 508 6.041 54.922 −4.680 1.00 35.35 C ATOM 90 CD2 TYR 508 5.389 53.946 −6.771 1.00 28.70 C ATOM 91 CE1 TYR 508 7.397 54.848 −5.054 1.00 36.51 C ATOM 92 CE2 TYR 508 6.730 53.862 −7.154 1.00 33.58 C ATOM 93 CZ TYR 508 7.735 54.309 −6.294 1.00 36.83 C ATOM 94 OH TYR 508 9.075 54.176 −6.649 1.00 40.00 O ATOM 95 N CYS 509 3.046 57.662 −5.717 1.00 28.11 N ATOM 96 CA CYS 509 2.593 58.516 −6.822 1.00 27.15 C ATOM 97 C CYS 509 3.509 58.310 −8.009 1.00 25.32 C ATOM 98 O CYS 509 4.717 58.512 −7.909 1.00 30.67 O ATOM 99 CB CYS 509 2.572 59.997 −6.464 1.00 25.77 C ATOM 100 SG CYS 509 1.888 61.004 −7.834 1.00 21.50 S ATOM 101 N SER 510 2.939 57.837 −9.102 1.00 23.50 N ATOM 102 CA SER 510 3.686 57.590 −10.321 1.00 25.91 C ATOM 103 C SER 510 3.052 58.414 −11.475 1.00 25.38 C ATOM 104 O SER 510 2.384 57.882 −12.364 1.00 32.01 O ATOM 105 CB SER 510 3.687 56.087 −10.612 1.00 24.85 C ATOM 106 OG SER 510 4.275 55.783 −11.867 1.00 29.60 O ATOM 107 N ARG 511 3.236 59.726 −11.434 1.00 19.35 N ATOM 108 CA ARG 511 2.689 60.596 −12.451 1.00 16.16 C ATOM 109 C ARG 511 3.757 61.589 −12.810 1.00 20.03 C ATOM 110 O ARG 511 4.825 61.584 −12.191 1.00 24.48 O ATOM 111 CB ARG 511 1.439 61.310 −11.952 1.00 13.51 C ATOM 112 CG ARG 511 0.300 60.358 −11.708 1.00 13.74 C ATOM 113 CD ARG 511 −0.994 61.084 −11.674 1.00 12.03 C ATOM 114 NE ARG 511 −2.111 60.169 −11.505 1.00 12.96 N ATOM 115 CZ ARG 511 −3.388 60.547 −11.582 1.00 16.84 C ATOM 116 NH1 ARG 511 −3.700 61.814 −11.841 1.00 18.71 N ATOM 117 NH2 ARG 511 −4.361 59.680 −11.342 1.00 18.41 N ATOM 118 N LEU 512 3.482 62.427 −13.810 1.00 16.95 N ATOM 119 CA LEU 512 4.457 63.400 −14.279 1.00 17.28 C ATOM 120 C LEU 512 4.337 64.645 −13.434 1.00 16.82 C ATOM 121 O LEU 512 3.544 65.534 −13.715 1.00 18.14 O ATOM 122 CB LEU 512 4.220 63.668 −15.756 1.00 18.43 C ATOM 123 CG LEU 512 4.357 62.372 −16.556 1.00 17.02 C ATOM 124 CD1 LEU 512 3.947 62.602 −17.993 1.00 15.13 C ATOM 125 CD2 LEU 512 5.796 61.865 −16.461 1.00 16.19 C ATOM 126 N LEU 513 5.226 64.748 −12.458 1.00 19.32 N ATOM 127 CA LEU 513 5.162 65.827 −11.501 1.00 18.08 C ATOM 128 C LEU 513 6.506 66.390 −11.089 1.00 15.12 C ATOM 129 O LEU 513 7.477 65.655 −10.948 1.00 14.11 O ATOM 130 CB LEU 513 4.454 65.273 −10.257 1.00 19.36 C ATOM 131 CG LEU 513 4.260 66.123 −9.012 1.00 20.52 C ATOM 132 CD1 LEU 513 3.338 67.259 −9.353 1.00 18.37 C ATOM 133 CD2 LEU 513 3.698 65.270 −7.902 1.00 12.16 C ATOM 134 N ASP 514 6.534 67.699 −10.853 1.00 16.07 N ATOM 135 CA ASP 514 7.735 68.384 −10.383 1.00 16.17 C ATOM 136 C ASP 514 7.257 68.946 −9.041 1.00 15.61 C ATOM 137 O ASP 514 6.480 69.890 −9.014 1.00 16.76 O ATOM 138 CB ASP 514 8.136 69.530 −11.327 1.00 15.99 C ATOM 139 CG ASP 514 9.016 69.076 −12.511 1.00 18.33 C ATOM 140 OD1 ASP 514 9.255 67.874 −12.736 1.00 17.22 O ATOM 141 OD2 ASP 514 9.492 69.961 −13.240 1.00 25.18 O ATOM 142 N LEU 515 7.660 68.338 −7.929 1.00 15.44 N ATOM 143 CA LEU 515 7.205 68.808 −6.623 1.00 14.64 C ATOM 144 C LEU 515 8.282 69.524 −5.802 1.00 14.01 C ATOM 145 O LEU 515 9.382 68.995 −5.603 1.00 8.40 O ATOM 146 CB LEU 515 6.613 67.625 −5.853 1.00 14.99 C ATOM 147 CG LEU 515 6.168 67.788 −4.396 1.00 9.59 C ATOM 148 CD1 LEU 515 5.054 68.775 −4.249 1.00 5.58 C ATOM 149 CD2 LEU 515 5.714 66.455 −3.924 1.00 8.85 C ATOM 150 N VAL 516 7.975 70.743 −5.355 1.00 10.50 N ATOM 151 CA VAL 516 8.927 71.514 −4.566 1.00 13.88 C ATOM 152 C VAL 516 8.504 71.601 −3.100 1.00 14.19 C ATOM 153 O VAL 516 7.400 72.059 −2.796 1.00 14.17 O ATOM 154 CB VAL 516 9.103 72.966 −5.111 1.00 15.97 C ATOM 155 CG1 VAL 516 10.134 73.711 −4.278 1.00 14.88 C ATOM 156 CG2 VAL 516 9.580 72.947 −6.570 1.00 14.16 C ATOM 157 N PHE 517 9.359 71.127 −2.191 1.00 17.00 N ATOM 158 CA PHE 517 9.072 71.205 −0.747 1.00 13.51 C ATOM 159 C PHE 517 9.776 72.469 −0.179 1.00 12.08 C ATOM 160 O PHE 517 11.009 72.587 −0.233 1.00 10.17 O ATOM 161 CB PHE 517 9.602 69.968 −0.024 1.00 15.54 C ATOM 162 CG PHE 517 8.940 68.662 −0.426 1.00 11.63 C ATOM 163 CD1 PHE 517 7.805 68.200 0.230 1.00 13.47 C ATOM 164 CD2 PHE 517 9.517 67.852 −1.400 1.00 9.53 C ATOM 165 CE1 PHE 517 7.256 66.952 −0.075 1.00 10.36 C ATOM 166 CE2 PHE 517 8.989 66.625 −1.703 1.00 8.49 C ATOM 167 CZ PHE 517 7.847 66.164 −1.036 1.00 10.76 C ATOM 168 N LEU 518 9.005 73.405 0.378 1.00 12.33 N ATOM 169 CA LEU 518 9.574 74.643 0.926 1.00 12.65 C ATOM 170 C LEU 518 9.420 74.659 2.453 1.00 11.22 C ATOM 171 O LEU 518 8.333 74.940 2.959 1.00 15.25 O ATOM 172 CB LEU 518 8.884 75.865 0.287 1.00 9.93 C ATOM 173 CG LEU 518 8.791 75.956 −1.244 1.00 8.05 C ATOM 174 CD1 LEU 518 7.840 77.076 −1.640 1.00 3.79 C ATOM 175 CD2 LEU 518 10.166 76.182 −1.857 1.00 10.27 C ATOM 176 N LEU 519 10.514 74.399 3.168 1.00 7.04 N ATOM 177 CA LEU 519 10.518 74.319 4.635 1.00 7.57 C ATOM 178 C LEU 519 10.795 75.567 5.440 1.00 4.28 C ATOM 179 O LEU 519 11.867 76.131 5.339 1.00 10.10 O ATOM 180 CB LEU 519 11.544 73.283 5.090 1.00 6.92 C ATOM 181 CG LEU 519 11.188 71.821 4.925 1.00 10.92 C ATOM 182 CD1 LEU 519 11.011 71.550 3.470 1.00 8.66 C ATOM 183 CD2 LEU 519 12.295 70.951 5.489 1.00 7.00 C ATOM 184 N ASP 520 9.891 75.908 6.352 1.00 11.69 N ATOM 185 CA ASP 520 10.051 77.081 7.217 1.00 13.58 C ATOM 186 C ASP 520 11.330 76.914 8.057 1.00 14.38 C ATOM 187 O ASP 520 11.469 75.933 8.775 1.00 18.86 O ATOM 188 CB ASP 520 8.813 77.201 8.116 1.00 15.76 C ATOM 189 CG ASP 520 8.737 78.528 8.839 1.00 14.91 C ATOM 190 OD1 ASP 520 9.744 78.970 9.394 1.00 15.53 O ATOM 191 OD2 ASP 520 7.671 79.143 8.870 1.00 15.00 O ATOM 192 N GLY 521 12.272 77.847 7.958 1.00 12.62 N ATOM 193 CA GLY 521 13.514 77.720 8.713 1.00 12.88 C ATOM 194 C GLY 521 13.617 78.637 9.919 1.00 17.26 C ATOM 195 O GLY 521 14.690 78.785 10.508 1.00 16.89 O ATOM 196 N SER 522 12.500 79.256 10.291 1.00 16.08 N ATOM 197 CA SER 522 12.452 80.168 11.420 1.00 17.98 C ATOM 198 C SER 522 12.484 79.424 12.750 1.00 18.91 C ATOM 199 O SER 522 12.359 78.197 12.792 1.00 17.50 O ATOM 200 CB SER 522 11.158 80.967 11.371 1.00 12.88 C ATOM 201 OG SER 522 10.069 80.128 11.708 1.00 12.60 O ATOM 202 N SER 523 12.547 80.206 13.828 1.00 17.68 N ATOM 203 CA SER 523 12.558 79.701 15.187 1.00 20.71 C ATOM 204 C SER 523 11.141 79.389 15.694 1.00 20.97 C ATOM 205 O SER 523 10.971 78.999 16.833 1.00 27.46 O ATOM 206 CB SER 523 13.253 80.693 16.111 1.00 21.32 C ATOM 207 OG SER 523 12.701 81.986 15.933 1.00 26.32 O ATOM 208 N ARG 524 10.117 79.590 14.872 1.00 18.19 N ATOM 209 CA ARG 524 8.765 79.240 15.278 1.00 19.04 C ATOM 210 C ARG 524 8.655 77.734 15.460 1.00 20.87 C ATOM 211 O ARG 524 7.724 77.235 16.106 1.00 22.79 O ATOM 212 CB ARG 524 7.758 79.709 14.246 1.00 20.78 C ATOM 213 CG ARG 524 7.654 81.183 14.225 1.00 22.74 C ATOM 214 CD ARG 524 7.449 81.671 15.627 1.00 23.78 C ATOM 215 NE ARG 524 7.265 83.107 15.661 1.00 29.27 N ATOM 216 CZ ARG 524 7.116 83.815 16.774 1.00 29.96 C ATOM 217 NH1 ARG 524 7.140 83.218 17.959 1.00 25.48 N ATOM 218 NH2 ARG 524 6.899 85.118 16.686 1.00 26.64 N ATOM 219 N LEU 525 9.561 77.014 14.804 1.00 19.12 N ATOM 220 CA LEU 525 9.645 75.571 14.915 1.00 17.29 C ATOM 221 C LEU 525 10.942 75.381 15.674 1.00 22.24 C ATOM 222 O LEU 525 11.950 76.041 15.374 1.00 21.49 O ATOM 223 CB LEU 525 9.817 74.913 13.546 1.00 19.88 C ATOM 224 CG LEU 525 8.695 74.845 12.523 1.00 15.92 C ATOM 225 CD1 LEU 525 9.236 74.241 11.253 1.00 11.38 C ATOM 226 CD2 LEU 525 7.556 73.989 13.080 1.00 16.42 C ATOM 227 N SER 526 10.912 74.519 16.682 1.00 23.55 N ATOM 228 CA SER 526 12.113 74.240 17.460 1.00 24.19 C ATOM 229 C SER 526 12.873 73.147 16.725 1.00 25.31 C ATOM 230 O SER 526 12.342 72.534 15.797 1.00 26.87 O ATOM 231 CB SER 526 11.737 73.746 18.855 1.00 22.27 C ATOM 232 OG SER 526 10.926 72.579 18.785 1.00 18.28 O ATOM 233 N GLU 527 14.093 72.868 17.160 1.00 26.48 N ATOM 234 CA GLU 527 14.900 71.829 16.539 1.00 26.18 C ATOM 235 C GLU 527 14.144 70.514 16.449 1.00 25.55 C ATOM 236 O GLU 527 14.134 69.859 15.406 1.00 30.55 O ATOM 237 CB GLU 527 16.172 71.620 17.349 1.00 28.26 C ATOM 238 CG GLU 527 17.025 70.471 16.880 1.00 37.14 C ATOM 239 CD GLU 527 18.417 70.909 16.459 1.00 44.14 C ATOM 240 OE1 GLU 527 19.082 71.637 17.238 1.00 44.73 O ATOM 241 OE2 GLU 527 18.853 70.506 15.351 1.00 47.92 O ATOM 242 N ALA 528 13.458 70.159 17.526 1.00 23.12 N ATOM 243 CA ALA 528 12.733 68.901 17.580 1.00 19.11 C ATOM 244 C ALA 528 11.547 68.875 16.650 1.00 19.81 C ATOM 245 O ALA 528 11.237 67.834 16.078 1.00 24.34 O ATOM 246 CB ALA 528 12.301 68.609 18.992 1.00 11.67 C ATOM 247 N GLU 529 10.873 70.015 16.518 1.00 19.26 N ATOM 248 CA GLU 529 9.697 70.126 15.659 1.00 19.04 C ATOM 249 C GLU 529 10.146 70.132 14.212 1.00 21.14 C ATOM 250 O GLU 529 9.491 69.543 13.358 1.00 26.65 O ATOM 251 CB GLU 529 8.908 71.403 15.974 1.00 17.25 C ATOM 252 CG GLU 529 8.144 71.355 17.292 1.00 12.54 C ATOM 253 CD GLU 529 7.447 72.663 17.624 1.00 18.73 C ATOM 254 OE1 GLU 529 8.120 73.725 17.576 1.00 16.59 O ATOM 255 OE2 GLU 529 6.236 72.623 17.966 1.00 18.64 O ATOM 256 N PHE 530 11.292 70.750 13.945 1.00 17.88 N ATOM 257 CA PHE 530 11.828 70.807 12.600 1.00 18.23 C ATOM 258 C PHE 530 12.139 69.383 12.147 1.00 21.70 C ATOM 259 O PHE 530 11.848 69.007 11.003 1.00 28.22 O ATOM 260 CB PHE 530 13.083 71.658 12.571 1.00 17.84 C ATOM 261 CG PHE 530 13.534 72.028 11.196 1.00 19.34 C ATOM 262 CD1 PHE 530 12.696 72.758 10.342 1.00 17.07 C ATOM 263 CD2 PHE 530 14.806 71.685 10.759 1.00 16.08 C ATOM 264 CE1 PHE 530 13.127 73.139 9.089 1.00 12.46 C ATOM 265 CE2 PHE 530 15.248 72.066 9.492 1.00 13.83 C ATOM 266 CZ PHE 530 14.413 72.789 8.664 1.00 11.80 C ATOM 267 N GLU 531 12.650 68.563 13.052 1.00 18.89 N ATOM 268 CA GLU 531 12.932 67.182 12.695 1.00 21.46 C ATOM 269 C GLU 531 11.660 66.478 12.263 1.00 17.63 C ATOM 270 O GLU 531 11.711 65.606 11.397 1.00 22.80 O ATOM 271 CB GLU 531 13.556 66.414 13.862 1.00 19.16 C ATOM 272 CG GLU 531 14.885 66.955 14.274 1.00 24.82 C ATOM 273 CD GLU 531 15.827 67.120 13.105 1.00 28.99 C ATOM 274 OE1 GLU 531 16.177 66.101 12.469 1.00 24.87 O ATOM 275 OE2 GLU 531 16.211 68.282 12.831 1.00 35.27 O ATOM 276 N VAL 532 10.529 66.840 12.864 1.00 15.71 N ATOM 277 CA VAL 532 9.257 66.211 12.509 1.00 15.96 C ATOM 278 C VAL 532 8.820 66.691 11.133 1.00 18.38 C ATOM 279 O VAL 532 8.234 65.921 10.357 1.00 22.15 O ATOM 280 CB VAL 532 8.149 66.493 13.551 1.00 15.96 C ATOM 281 CG1 VAL 532 6.825 65.847 13.117 1.00 6.01 C ATOM 282 CG2 VAL 532 8.575 65.950 14.908 1.00 13.74 C ATOM 283 N LEU 533 9.150 67.943 10.821 1.00 14.73 N ATOM 284 CA LEU 533 8.826 68.530 9.536 1.00 14.97 C ATOM 285 C LEU 533 9.602 67.771 8.476 1.00 16.19 C ATOM 286 O LEU 533 9.044 67.348 7.458 1.00 18.03 O ATOM 287 CB LEU 533 9.243 70.008 9.496 1.00 14.45 C ATOM 288 CG LEU 533 8.780 70.717 8.220 1.00 12.15 C ATOM 289 CD1 LEU 533 7.283 70.472 8.038 1.00 14.73 C ATOM 290 CD2 LEU 533 9.060 72.183 8.282 1.00 8.85 C ATOM 291 N LYS 534 10.899 67.640 8.704 1.00 12.22 N ATOM 292 CA LYS 534 11.766 66.936 7.785 1.00 13.88 C ATOM 293 C LYS 534 11.337 65.492 7.592 1.00 13.67 C ATOM 294 O LYS 534 11.356 64.974 6.478 1.00 19.68 O ATOM 295 CB LYS 534 13.210 67.016 8.282 1.00 17.43 C ATOM 296 CG LYS 534 13.819 68.393 8.100 1.00 13.24 C ATOM 297 CD LYS 534 15.002 68.635 9.036 1.00 18.41 C ATOM 298 CE LYS 534 15.936 67.455 9.114 1.00 20.81 C ATOM 299 NZ LYS 534 17.017 67.696 10.101 1.00 18.52 N ATOM 300 N ALA 535 10.871 64.858 8.656 1.00 13.06 N ATOM 301 CA ALA 535 10.442 63.472 8.563 1.00 9.85 C ATOM 302 C ALA 535 9.164 63.382 7.745 1.00 13.30 C ATOM 303 O ALA 535 8.911 62.361 7.082 1.00 15.28 O ATOM 304 CB ALA 535 10.254 62.901 9.923 1.00 8.53 C ATOM 305 N PHE 536 8.348 64.428 7.797 1.00 13.50 N ATOM 306 CA PHE 536 7.117 64.490 6.996 1.00 11.73 C ATOM 307 C PHE 536 7.537 64.558 5.521 1.00 14.75 C ATOM 308 O PHE 536 6.962 63.887 4.654 1.00 17.83 O ATOM 309 CB PHE 536 6.369 65.756 7.350 1.00 6.75 C ATOM 310 CG PHE 536 5.252 66.076 6.419 1.00 12.43 C ATOM 311 CD1 PHE 536 4.113 65.288 6.388 1.00 13.94 C ATOM 312 CD2 PHE 536 5.314 67.191 5.585 1.00 10.83 C ATOM 313 CE1 PHE 536 3.040 65.604 5.534 1.00 15.05 C ATOM 314 CE2 PHE 536 4.260 67.511 4.736 1.00 11.68 C ATOM 315 CZ PHE 536 3.114 66.720 4.711 1.00 9.74 C ATOM 316 N VAL 537 8.550 65.375 5.250 1.00 13.66 N ATOM 317 CA VAL 537 9.068 65.549 3.903 1.00 14.58 C ATOM 318 C VAL 537 9.593 64.209 3.418 1.00 16.40 C ATOM 319 O VAL 537 9.176 63.735 2.376 1.00 21.04 O ATOM 320 CB VAL 537 10.143 66.684 3.858 1.00 12.13 C ATOM 321 CG1 VAL 537 10.864 66.706 2.527 1.00 7.80 C ATOM 322 CG2 VAL 537 9.459 68.040 4.070 1.00 2.11 C ATOM 323 N VAL 538 10.397 63.541 4.231 1.00 17.90 N ATOM 324 CA VAL 538 10.929 62.228 3.867 1.00 15.91 C ATOM 325 C VAL 538 9.842 61.187 3.559 1.00 17.55 C ATOM 326 O VAL 538 9.946 60.491 2.538 1.00 15.35 O ATOM 327 CB VAL 538 11.893 61.693 4.948 1.00 17.65 C ATOM 328 CG1 VAL 538 12.129 60.169 4.791 1.00 9.43 C ATOM 329 CG2 VAL 538 13.200 62.447 4.849 1.00 9.34 C ATOM 330 N ASP 539 8.818 61.040 4.410 1.00 17.17 N ATOM 331 CA ASP 539 7.798 60.050 4.068 1.00 21.22 C ATOM 332 C ASP 539 6.892 60.418 2.919 1.00 17.63 C ATOM 333 O ASP 539 6.313 59.526 2.305 1.00 20.04 O ATOM 334 CB ASP 539 7.048 59.412 5.257 1.00 29.14 C ATOM 335 CG ASP 539 6.836 60.345 6.384 1.00 35.47 C ATOM 336 OD1 ASP 539 6.283 61.428 6.128 1.00 44.75 O ATOM 337 OD2 ASP 539 7.219 59.993 7.523 1.00 36.63 O ATOM 338 N MET 540 6.810 61.709 2.601 1.00 16.39 N ATOM 339 CA MET 540 6.063 62.172 1.428 1.00 13.96 C ATOM 340 C MET 540 6.857 61.593 0.269 1.00 10.86 C ATOM 341 O MET 540 6.333 60.862 −0.539 1.00 14.71 O ATOM 342 CB MET 540 6.159 63.688 1.294 1.00 22.58 C ATOM 343 CG MET 540 5.149 64.497 2.065 1.00 31.44 C ATOM 344 SD MET 540 3.503 64.541 1.326 1.00 45.37 S ATOM 345 CE MET 540 3.679 65.729 0.060 1.00 31.22 C ATOM 346 N MET 541 8.163 61.870 0.261 1.00 13.18 N ATOM 347 CA MET 541 9.116 61.398 −0.751 1.00 10.35 C ATOM 348 C MET 541 9.107 59.895 −0.969 1.00 16.49 C ATOM 349 O MET 541 9.225 59.438 −2.111 1.00 20.40 O ATOM 350 CB MET 541 10.537 61.813 −0.370 1.00 9.58 C ATOM 351 CG MET 541 10.820 63.276 −0.620 1.00 11.21 C ATOM 352 SD MET 541 12.434 63.842 −0.058 1.00 12.89 S ATOM 353 CE MET 541 13.392 63.835 −1.500 1.00 15.28 C ATOM 354 N GLU 542 8.985 59.119 0.107 1.00 16.56 N ATOM 355 CA GLU 542 8.971 57.657 −0.008 1.00 13.92 C ATOM 356 C GLU 542 7.759 57.174 −0.818 1.00 14.26 C ATOM 357 O GLU 542 7.789 56.104 −1.430 1.00 15.31 O ATOM 358 CB GLU 542 8.925 57.018 1.382 1.00 12.65 C ATOM 359 CG GLU 542 10.175 57.228 2.230 1.00 15.01 C ATOM 360 CD GLU 542 10.155 56.422 3.528 1.00 13.47 C ATOM 361 OE1 GLU 542 9.181 55.694 3.785 1.00 18.27 O ATOM 362 OE2 GLU 542 11.109 56.518 4.308 1.00 15.89 O ATOM 363 N ARG 543 6.688 57.961 −0.789 1.00 12.61 N ATOM 364 CA ARG 543 5.451 57.633 −1.473 1.00 10.82 C ATOM 365 C ARG 543 5.388 58.327 −2.827 1.00 11.37 C ATOM 366 O ARG 543 4.310 58.574 −3.373 1.00 13.23 O ATOM 367 CB ARG 543 4.285 58.069 −0.593 1.00 9.75 C ATOM 368 CG ARG 543 4.168 57.203 0.632 1.00 8.93 C ATOM 369 CD ARG 543 3.353 57.841 1.737 1.00 9.66 C ATOM 370 NE ARG 543 3.241 56.878 2.835 1.00 17.05 N ATOM 371 CZ ARG 543 4.196 56.652 3.737 1.00 12.00 C ATOM 372 NH1 ARG 543 5.341 57.332 3.697 1.00 9.90 N ATOM 373 NH2 ARG 543 4.044 55.681 4.634 1.00 15.47 N ATOM 374 N LEU 544 6.554 58.559 −3.401 1.00 13.34 N ATOM 375 CA LEU 544 6.676 59.214 −4.688 1.00 15.24 C ATOM 376 C LEU 544 7.606 58.388 −5.574 1.00 16.79 C ATOM 377 O LEU 544 8.633 57.871 −5.103 1.00 17.01 O ATOM 378 CB LEU 544 7.318 60.578 −4.465 1.00 12.06 C ATOM 379 CG LEU 544 6.539 61.886 −4.546 1.00 11.78 C ATOM 380 CD1 LEU 544 5.138 61.769 −4.046 1.00 11.41 C ATOM 381 CD2 LEU 544 7.315 62.896 −3.751 1.00 8.97 C ATOM 382 N ARG 545 7.283 58.269 −6.855 1.00 15.28 N ATOM 383 CA ARG 545 8.187 57.551 −7.740 1.00 15.08 C ATOM 384 C ARG 545 9.110 58.627 −8.278 1.00 17.33 C ATOM 385 O ARG 545 8.794 59.266 −9.283 1.00 19.41 O ATOM 386 CB ARG 545 7.431 56.887 −8.875 1.00 16.99 C ATOM 387 CG ARG 545 8.327 56.142 −9.837 1.00 20.50 C ATOM 388 CD ARG 545 7.453 55.493 −10.868 1.00 26.46 C ATOM 389 NE ARG 545 8.195 54.796 −11.903 1.00 28.51 N ATOM 390 CZ ARG 545 7.644 53.877 −12.690 1.00 33.26 C ATOM 391 NH1 ARG 545 6.357 53.572 −12.541 1.00 31.05 N ATOM 392 NH2 ARG 545 8.368 53.259 −13.617 1.00 30.37 N ATOM 393 N ILE 546 10.205 58.879 −7.556 1.00 18.27 N ATOM 394 CA ILE 546 11.177 59.909 −7.919 1.00 18.03 C ATOM 395 C ILE 546 12.039 59.480 −9.084 1.00 19.30 C ATOM 396 O ILE 546 12.750 58.477 −9.014 1.00 20.66 O ATOM 397 CB ILE 546 12.065 60.290 −6.714 1.00 13.91 C ATOM 398 CG1 ILE 546 11.234 60.956 −5.622 1.00 10.37 C ATOM 399 CG2 ILE 546 13.134 61.243 −7.134 1.00 10.93 C ATOM 400 CD1 ILE 546 11.981 61.088 −4.306 1.00 8.23 C ATOM 401 N SER 547 11.986 60.270 −10.152 1.00 20.13 N ATOM 402 CA SER 547 12.725 59.968 −11.360 1.00 18.38 C ATOM 403 C SER 547 12.518 61.108 −12.348 1.00 20.11 C ATOM 404 O SER 547 11.443 61.710 −12.407 1.00 19.86 O ATOM 405 CB SER 547 12.191 58.664 −11.956 1.00 19.67 C ATOM 406 OG SER 547 12.939 58.238 −13.078 1.00 24.56 O ATOM 407 N GLN 548 13.542 61.364 −13.156 1.00 21.04 N ATOM 408 CA GLN 548 13.527 62.426 −14.159 1.00 22.05 C ATOM 409 C GLN 548 12.335 62.253 −15.121 1.00 23.36 C ATOM 410 O GLN 548 11.802 63.236 −15.645 1.00 26.68 O ATOM 411 CB GLN 548 14.869 62.399 −14.905 1.00 23.09 C ATOM 412 CG GLN 548 15.601 63.727 −15.086 1.00 25.87 C ATOM 413 CD GLN 548 15.738 64.560 −13.825 1.00 27.98 C ATOM 414 OE1 GLN 548 15.630 65.783 −13.890 1.00 34.12 O ATOM 415 NE2 GLN 548 15.977 63.925 −12.684 1.00 25.55 N ATOM 416 N LYS 549 11.894 61.009 −15.304 1.00 21.82 N ATOM 417 CA LYS 549 10.766 60.701 −16.182 1.00 21.44 C ATOM 418 C LYS 549 9.453 60.554 −15.390 1.00 22.62 C ATOM 419 O LYS 549 8.374 60.330 −15.971 1.00 18.28 O ATOM 420 CB LYS 549 11.041 59.406 −16.955 1.00 24.39 C ATOM 421 CG LYS 549 12.431 59.320 −17.575 1.00 34.15 C ATOM 422 CD LYS 549 12.762 60.573 −18.390 1.00 44.91 C ATOM 423 CE LYS 549 14.154 60.513 −19.044 1.00 51.62 C ATOM 424 NZ LYS 549 15.302 60.529 −18.076 1.00 53.07 N ATOM 425 N TRP 550 9.535 60.665 −14.064 1.00 19.15 N ATOM 426 CA TRP 550 8.350 60.544 −13.241 1.00 18.15 C ATOM 427 C TRP 550 8.178 61.740 −12.313 1.00 17.14 C ATOM 428 O TRP 550 7.755 62.808 −12.759 1.00 17.54 O ATOM 429 CB TRP 550 8.383 59.219 −12.473 1.00 22.31 C ATOM 430 CG TRP 550 8.360 58.034 −13.381 1.00 24.07 C ATOM 431 CD1 TRP 550 9.420 57.250 −13.752 1.00 25.74 C ATOM 432 CD2 TRP 550 7.228 57.533 −14.096 1.00 23.63 C ATOM 433 NE1 TRP 550 9.016 56.302 −14.667 1.00 25.59 N ATOM 434 CE2 TRP 550 7.665 56.461 −14.894 1.00 25.02 C ATOM 435 CE3 TRP 550 5.875 57.905 −14.136 1.00 23.56 C ATOM 436 CZ2 TRP 550 6.807 55.743 −15.734 1.00 25.72 C ATOM 437 CZ3 TRP 550 5.017 57.196 −14.970 1.00 25.40 C ATOM 438 CH2 TRP 550 5.487 56.125 −15.758 1.00 24.34 C ATOM 439 N VAL 551 8.465 61.572 −11.022 1.00 18.80 N ATOM 440 CA VAL 551 8.321 62.674 −10.069 1.00 16.63 C ATOM 441 C VAL 551 9.695 63.240 −9.739 1.00 14.99 C ATOM 442 O VAL 551 10.599 62.501 −9.362 1.00 15.74 O ATOM 443 CB VAL 551 7.614 62.214 −8.731 1.00 16.18 C ATOM 444 CG1 VAL 551 7.477 63.406 −7.761 1.00 10.00 C ATOM 445 CG2 VAL 551 6.231 61.611 −9.016 1.00 12.35 C ATOM 446 N ARG 552 9.876 64.536 −9.950 1.00 14.51 N ATOM 447 CA ARG 552 11.142 65.180 −9.612 1.00 14.95 C ATOM 448 C ARG 552 10.860 66.010 −8.358 1.00 14.50 C ATOM 449 O ARG 552 9.756 66.543 −8.188 1.00 12.82 O ATOM 450 CB ARG 552 11.639 66.066 −10.757 1.00 13.60 C ATOM 451 CG ARG 552 11.890 65.318 −12.052 1.00 14.38 C ATOM 452 CD ARG 552 12.686 66.146 −13.018 1.00 14.42 C ATOM 453 NE ARG 552 11.980 67.368 −13.388 1.00 22.75 N ATOM 454 CZ ARG 552 12.544 68.383 −14.035 1.00 19.53 C ATOM 455 NH1 ARG 552 13.824 68.318 −14.381 1.00 24.67 N ATOM 456 NH2 ARG 552 11.823 69.436 −14.383 1.00 12.20 N ATOM 457 N VAL 553 11.844 66.108 −7.469 1.00 18.50 N ATOM 458 CA VAL 553 11.654 66.840 −6.209 1.00 19.75 C ATOM 459 C VAL 553 12.740 67.861 −5.972 1.00 19.66 C ATOM 460 O VAL 553 13.872 67.695 −6.406 1.00 22.27 O ATOM 461 CB VAL 553 11.677 65.886 −4.956 1.00 18.81 C ATOM 462 CG1 VAL 553 10.441 64.952 −4.920 1.00 7.54 C ATOM 463 CG2 VAL 553 12.966 65.070 −4.942 1.00 19.00 C ATOM 464 N ALA 554 12.385 68.930 −5.279 1.00 17.64 N ATOM 465 CA ALA 554 13.356 69.958 −4.924 1.00 16.45 C ATOM 466 C ALA 554 13.038 70.208 −3.450 1.00 14.99 C ATOM 467 O ALA 554 11.882 70.006 −3.030 1.00 12.08 O ATOM 468 CB ALA 554 13.153 71.224 −5.764 1.00 6.96 C ATOM 469 N VAL 555 14.063 70.516 −2.651 1.00 13.54 N ATOM 470 CA VAL 555 13.879 70.778 −1.220 1.00 14.53 C ATOM 471 C VAL 555 14.590 72.068 −0.892 1.00 11.41 C ATOM 472 O VAL 555 15.769 72.191 −1.175 1.00 10.57 O ATOM 473 CB VAL 555 14.402 69.594 −0.318 1.00 14.50 C ATOM 474 CG1 VAL 555 14.355 69.980 1.182 1.00 10.37 C ATOM 475 CG2 VAL 555 13.491 68.358 −0.515 1.00 11.88 C ATOM 476 N VAL 556 13.857 73.044 −0.359 1.00 11.91 N ATOM 477 CA VAL 556 14.424 74.337 −0.026 1.00 14.48 C ATOM 478 C VAL 556 13.996 74.729 1.394 1.00 18.03 C ATOM 479 O VAL 556 12.807 74.771 1.717 1.00 18.24 O ATOM 480 CB VAL 556 13.929 75.435 −0.999 1.00 16.63 C ATOM 481 CG1 VAL 556 14.721 76.700 −0.812 1.00 12.12 C ATOM 482 CG2 VAL 556 14.022 74.948 −2.450 1.00 17.97 C ATOM 483 N GLU 557 14.977 75.023 2.228 1.00 15.76 N ATOM 484 CA GLU 557 14.750 75.431 3.594 1.00 16.18 C ATOM 485 C GLU 557 14.810 76.937 3.456 1.00 19.44 C ATOM 486 O GLU 557 15.751 77.448 2.847 1.00 20.78 O ATOM 487 CB GLU 557 15.922 74.921 4.451 1.00 21.73 C ATOM 488 CG GLU 557 16.042 75.479 5.890 1.00 22.98 C ATOM 489 CD GLU 557 17.093 76.581 6.011 1.00 26.66 C ATOM 490 OE1 GLU 557 18.322 76.271 6.047 1.00 27.04 O ATOM 491 OE2 GLU 557 16.666 77.755 6.075 1.00 18.13 O ATOM 492 N TYR 558 13.823 77.668 3.964 1.00 16.82 N ATOM 493 CA TYR 558 13.878 79.117 3.826 1.00 19.30 C ATOM 494 C TYR 558 13.894 79.898 5.123 1.00 21.56 C ATOM 495 O TYR 558 13.367 79.450 6.146 1.00 18.80 O ATOM 496 CB TYR 558 12.769 79.638 2.903 1.00 18.20 C ATOM 497 CG TYR 558 11.350 79.457 3.414 1.00 21.51 C ATOM 498 CD1 TYR 558 10.681 78.250 3.251 1.00 19.46 C ATOM 499 CD2 TYR 558 10.662 80.511 4.037 1.00 17.80 C ATOM 500 CE1 TYR 558 9.360 78.101 3.689 1.00 17.32 C ATOM 501 CE2 TYR 558 9.361 80.361 4.462 1.00 11.71 C ATOM 502 CZ TYR 558 8.727 79.158 4.289 1.00 14.15 C ATOM 503 OH TYR 558 7.443 78.997 4.727 1.00 21.33 O ATOM 504 N HIS 559 14.548 81.054 5.077 1.00 22.82 N ATOM 505 CA HIS 559 14.659 81.942 6.228 1.00 22.57 C ATOM 506 C HIS 559 14.761 83.343 5.632 1.00 24.28 C ATOM 507 O HIS 559 13.803 83.820 5.035 1.00 24.51 O ATOM 508 CB HIS 559 15.877 81.580 7.084 1.00 23.05 C ATOM 509 CG HIS 559 17.138 81.409 6.295 1.00 31.12 C ATOM 510 ND1 HIS 559 17.327 80.384 5.400 1.00 35.63 N ATOM 511 CD2 HIS 559 18.274 82.154 6.254 1.00 35.29 C ATOM 512 CE1 HIS 559 18.512 80.499 4.841 1.00 34.53 C ATOM 513 NE2 HIS 559 19.109 81.564 5.342 1.00 33.84 N ATOM 514 N ASP 560 15.895 84.013 5.788 1.00 29.85 N ATOM 515 CA ASP 560 16.077 85.336 5.194 1.00 37.48 C ATOM 516 C ASP 560 16.114 85.132 3.676 1.00 36.28 C ATOM 517 O ASP 560 15.522 85.892 2.912 1.00 39.28 O ATOM 518 CB ASP 560 17.391 85.957 5.686 1.00 46.15 C ATOM 519 CG ASP 560 17.242 87.430 6.059 1.00 55.52 C ATOM 520 OD1 ASP 560 16.086 87.905 6.195 1.00 60.53 O ATOM 521 OD2 ASP 560 18.286 88.112 6.218 1.00 59.22 O ATOM 522 N GLY 561 16.849 84.101 3.271 1.00 34.51 N ATOM 523 CA GLY 561 16.973 83.729 1.879 1.00 29.45 C ATOM 524 C GLY 561 16.599 82.261 1.769 1.00 28.57 C ATOM 525 O GLY 561 15.991 81.698 2.689 1.00 25.19 O ATOM 526 N SER 562 17.044 81.614 0.700 1.00 27.35 N ATOM 527 CA SER 562 16.730 80.216 0.502 1.00 26.74 C ATOM 528 C SER 562 17.984 79.376 0.408 1.00 28.35 C ATOM 529 O SER 562 18.981 79.803 −0.164 1.00 29.74 O ATOM 530 CB SER 562 15.903 80.028 −0.770 1.00 25.65 C ATOM 531 OG SER 562 14.733 80.834 −0.746 1.00 25.69 O ATOM 532 N HIS 563 17.952 78.221 1.058 1.00 26.61 N ATOM 533 CA HIS 563 19.052 77.273 1.016 1.00 28.05 C ATOM 534 C HIS 563 18.452 76.026 0.354 1.00 25.45 C ATOM 535 O HIS 563 17.662 75.317 0.977 1.00 26.54 O ATOM 536 CB HIS 563 19.565 76.923 2.425 1.00 33.01 C ATOM 537 CG HIS 563 20.592 77.879 2.971 1.00 42.56 C ATOM 538 ND1 HIS 563 21.368 77.586 4.081 1.00 41.44 N ATOM 539 CD2 HIS 563 20.950 79.129 2.588 1.00 41.45 C ATOM 540 CE1 HIS 563 22.148 78.615 4.351 1.00 39.61 C ATOM 541 NE2 HIS 563 21.919 79.566 3.466 1.00 38.40 N ATOM 542 N ALA 564 18.736 75.824 −0.929 1.00 22.09 N ATOM 543 CA ALA 564 18.220 74.671 −1.653 1.00 20.28 C ATOM 544 C ALA 564 19.183 73.513 −1.505 1.00 18.53 C ATOM 545 O ALA 564 20.359 73.650 −1.785 1.00 21.05 O ATOM 546 CB ALA 564 18.040 75.005 −3.108 1.00 16.92 C ATOM 547 N TYR 565 18.675 72.384 −1.028 1.00 18.56 N ATOM 548 CA TYR 565 19.463 71.183 −0.840 1.00 15.69 C ATOM 549 C TYR 565 19.236 70.245 −2.021 1.00 16.43 C ATOM 550 O TYR 565 20.096 69.461 −2.376 1.00 14.38 O ATOM 551 CB TYR 565 19.035 70.473 0.434 1.00 18.28 C ATOM 552 CG TYR 565 19.373 71.214 1.691 1.00 20.29 C ATOM 553 CD1 TYR 565 18.609 72.296 2.105 1.00 19.07 C ATOM 554 CD2 TYR 565 20.450 70.816 2.485 1.00 24.63 C ATOM 555 CE1 TYR 565 18.899 72.966 3.277 1.00 23.97 C ATOM 556 CE2 TYR 565 20.752 71.476 3.670 1.00 25.91 C ATOM 557 CZ TYR 565 19.971 72.552 4.061 1.00 24.70 C ATOM 558 OH TYR 565 20.247 73.212 5.240 1.00 28.07 O ATOM 559 N ILE 566 18.050 70.302 −2.599 1.00 16.43 N ATOM 560 CA ILE 566 17.720 69.445 −3.721 1.00 16.83 C ATOM 561 C ILE 566 17.121 70.236 −4.876 1.00 14.70 C ATOM 562 O ILE 566 16.222 71.044 −4.662 1.00 16.54 O ATOM 563 CB ILE 566 16.690 68.368 −3.301 1.00 15.97 C ATOM 564 CG1 ILE 566 17.187 67.570 −2.097 1.00 18.44 C ATOM 565 CG2 ILE 566 16.392 67.432 −4.446 1.00 16.90 C ATOM 566 CD1 ILE 566 18.416 66.752 −2.370 1.00 28.29 C ATOM 567 N GLY 567 17.667 70.052 −6.075 1.00 14.70 N ATOM 568 CA GLY 567 17.123 70.694 −7.261 1.00 14.69 C ATOM 569 C GLY 567 16.372 69.660 −8.106 1.00 15.85 C ATOM 570 O GLY 567 16.730 68.469 −8.109 1.00 16.56 O ATOM 571 N LEU 568 15.392 70.097 −8.892 1.00 17.74 N ATOM 572 CA LEU 568 14.622 69.174 −9.722 1.00 16.77 C ATOM 573 C LEU 568 15.471 68.292 −10.628 1.00 18.54 C ATOM 574 O LEU 568 15.177 67.117 −10.807 1.00 24.33 O ATOM 575 CB LEU 568 13.591 69.935 −10.559 1.00 11.30 C ATOM 576 CG LEU 568 12.456 70.603 −9.767 1.00 11.42 C ATOM 577 CD1 LEU 568 11.744 71.638 −10.640 1.00 6.22 C ATOM 578 CD2 LEU 568 11.508 69.583 −9.227 1.00 3.91 C ATOM 579 N LYS 569 16.512 68.864 −11.212 1.00 23.19 N ATOM 580 CA LYS 569 17.393 68.123 −12.124 1.00 23.15 C ATOM 581 C LYS 569 18.459 67.248 −11.458 1.00 23.97 C ATOM 582 O LYS 569 19.272 66.623 −12.149 1.00 21.55 O ATOM 583 CB LYS 569 18.087 69.098 −13.067 1.00 23.83 C ATOM 584 CG LYS 569 17.154 69.828 −14.027 1.00 29.42 C ATOM 585 CD LYS 569 17.882 71.015 −14.638 1.00 32.89 C ATOM 586 CE LYS 569 16.994 71.782 −15.588 1.00 36.32 C ATOM 587 NZ LYS 569 17.497 71.604 −16.981 1.00 42.82 N ATOM 588 N ASP 570 18.477 67.207 −10.128 1.00 24.14 N ATOM 589 CA ASP 570 19.476 66.409 −9.435 1.00 26.34 C ATOM 590 C ASP 570 19.300 64.950 −9.749 1.00 31.25 C ATOM 591 O ASP 570 18.335 64.336 −9.314 1.00 34.28 O ATOM 592 CB ASP 570 19.389 66.624 −7.930 1.00 23.64 C ATOM 593 CG ASP 570 20.081 67.890 −7.493 1.00 24.59 C ATOM 594 OD1 ASP 570 20.678 68.575 −8.352 1.00 27.05 O ATOM 595 OD2 ASP 570 20.041 68.200 −6.289 1.00 24.49 O ATOM 596 N ARG 571 20.223 64.390 −10.523 1.00 34.80 N ATOM 597 CA ARG 571 20.126 62.983 −10.869 1.00 35.48 C ATOM 598 C ARG 571 20.829 62.155 −9.817 1.00 30.97 C ATOM 599 O ARG 571 22.025 61.861 −9.922 1.00 29.82 O ATOM 600 CB ARG 571 20.710 62.721 −12.246 1.00 46.16 C ATOM 601 CG ARG 571 20.453 61.315 −12.743 1.00 60.41 C ATOM 602 CD ARG 571 21.501 60.929 −13.762 1.00 70.06 C ATOM 603 NE ARG 571 21.379 59.540 −14.198 1.00 76.92 N ATOM 604 CZ ARG 571 22.061 59.032 −15.218 1.00 82.89 C ATOM 605 NH1 ARG 571 22.910 59.802 −15.892 1.00 86.30 N ATOM 606 NH2 ARG 571 21.877 57.770 −15.579 1.00 85.49 N ATOM 607 N LYS 572 20.093 61.876 −8.750 1.00 23.93 N ATOM 608 CA LYS 572 20.588 61.086 −7.634 1.00 20.81 C ATOM 609 C LYS 572 19.511 60.067 −7.365 1.00 16.06 C ATOM 610 O LYS 572 18.381 60.237 −7.789 1.00 17.31 O ATOM 611 CB LYS 572 20.843 61.966 −6.393 1.00 18.06 C ATOM 612 CG LYS 572 22.004 62.929 −6.578 1.00 20.31 C ATOM 613 CD LYS 572 22.138 63.949 −5.468 1.00 23.14 C ATOM 614 CE LYS 572 23.202 64.988 −5.836 1.00 24.18 C ATOM 615 NZ LYS 572 23.545 65.977 −4.755 1.00 20.57 N ATOM 616 N ARG 573 19.857 58.956 −6.750 1.00 20.74 N ATOM 617 CA ARG 573 18.834 57.949 −6.475 1.00 22.56 C ATOM 618 C ARG 573 17.883 58.457 −5.379 1.00 20.74 C ATOM 619 O ARG 573 18.280 59.311 −4.565 1.00 23.29 O ATOM 620 CB ARG 573 19.498 56.617 −6.138 1.00 27.37 C ATOM 621 CG ARG 573 20.682 56.741 −5.228 1.00 35.56 C ATOM 622 CD ARG 573 21.369 55.399 −5.040 1.00 44.36 C ATOM 623 NE ARG 573 22.340 55.095 −6.087 1.00 49.02 N ATOM 624 CZ ARG 573 22.261 54.052 −6.916 1.00 54.28 C ATOM 625 NH1 ARG 573 21.240 53.191 −6.847 1.00 56.03 N ATOM 626 NH2 ARG 573 23.226 53.851 −7.805 1.00 52.89 N ATOM 627 N PRO 574 16.610 57.997 −5.375 1.00 17.46 N ATOM 628 CA PRO 574 15.610 58.412 −4.392 1.00 12.01 C ATOM 629 C PRO 574 16.092 58.327 −2.953 1.00 14.44 C ATOM 630 O PRO 574 15.816 59.220 −2.163 1.00 17.36 O ATOM 631 CB PRO 574 14.472 57.442 −4.650 1.00 16.28 C ATOM 632 CG PRO 574 14.547 57.219 −6.088 1.00 11.38 C ATOM 633 CD PRO 574 16.024 57.002 −6.291 1.00 18.26 C ATOM 634 N SER 575 16.880 57.305 −2.639 1.00 13.89 N ATOM 635 CA SER 575 17.394 57.104 −1.289 1.00 15.54 C ATOM 636 C SER 575 18.385 58.170 −0.880 1.00 18.01 C ATOM 637 O SER 575 18.394 58.619 0.269 1.00 19.91 O ATOM 638 CB SER 575 18.052 55.742 −1.163 1.00 11.08 C ATOM 639 OG SER 575 19.038 55.601 −2.157 1.00 15.54 O ATOM 640 N GLU 576 19.221 58.599 −1.810 1.00 19.82 N ATOM 641 CA GLU 576 20.180 59.616 −1.445 1.00 19.22 C ATOM 642 C GLU 576 19.456 60.923 −1.244 1.00 19.69 C ATOM 643 O GLU 576 19.754 61.678 −0.322 1.00 21.30 O ATOM 644 CB GLU 576 21.287 59.774 −2.483 1.00 24.50 C ATOM 645 CG GLU 576 22.414 60.647 −1.930 1.00 34.07 C ATOM 646 CD GLU 576 23.375 61.108 −2.979 1.00 44.16 C ATOM 647 OE1 GLU 576 23.681 60.305 −3.893 1.00 48.95 O ATOM 648 OE2 GLU 576 23.816 62.283 −2.891 1.00 50.40 O ATOM 649 N LEU 577 18.495 61.191 −2.106 1.00 18.39 N ATOM 650 CA LEU 577 17.734 62.419 −1.993 1.00 18.06 C ATOM 651 C LEU 577 17.042 62.469 −0.640 1.00 15.66 C ATOM 652 O LEU 577 17.081 63.482 0.056 1.00 16.14 O ATOM 653 CB LEU 577 16.715 62.518 −3.141 1.00 14.46 C ATOM 654 CG LEU 577 17.362 62.681 −4.522 1.00 13.01 C ATOM 655 CD1 LEU 577 16.309 62.686 −5.592 1.00 12.16 C ATOM 656 CD2 LEU 577 18.182 63.963 −4.564 1.00 10.44 C ATOM 657 N ARG 578 16.431 61.356 −0.258 1.00 18.03 N ATOM 658 CA ARG 578 15.721 61.282 1.013 1.00 16.42 C ATOM 659 C ARG 578 16.656 61.533 2.177 1.00 18.59 C ATOM 660 O ARG 578 16.276 62.207 3.136 1.00 23.30 O ATOM 661 CB ARG 578 15.005 59.939 1.169 1.00 14.49 C ATOM 662 CG ARG 578 13.822 59.783 0.220 1.00 9.10 C ATOM 663 CD ARG 578 12.924 58.631 0.583 1.00 7.18 C ATOM 664 NE ARG 578 13.668 57.391 0.758 1.00 13.10 N ATOM 665 CZ ARG 578 13.780 56.455 −0.164 1.00 11.87 C ATOM 666 NH1 ARG 578 13.191 56.622 −1.340 1.00 9.76 N ATOM 667 NH2 ARG 578 14.501 55.368 0.083 1.00 14.50 N ATOM 668 N ARG 579 17.889 61.046 2.070 1.00 17.36 N ATOM 669 CA ARG 579 18.879 61.248 3.119 1.00 17.76 C ATOM 670 C ARG 579 19.246 62.719 3.237 1.00 18.99 C ATOM 671 O ARG 579 19.372 63.252 4.341 1.00 21.54 O ATOM 672 CB ARG 579 20.132 60.414 2.833 1.00 18.77 C ATOM 673 CG ARG 579 21.347 60.787 3.684 1.00 24.17 C ATOM 674 CD ARG 579 22.532 59.857 3.461 1.00 26.24 C ATOM 675 NE ARG 579 22.856 59.678 2.045 1.00 36.84 N ATOM 676 CZ ARG 579 22.428 58.654 1.296 1.00 39.67 C ATOM 677 NH1 ARG 579 21.640 57.703 1.800 1.00 37.99 N ATOM 678 NH2 ARG 579 22.861 58.521 0.052 1.00 40.49 N ATOM 679 N ILE 580 19.433 63.377 2.101 1.00 18.54 N ATOM 680 CA ILE 580 19.784 64.790 2.098 1.00 14.36 C ATOM 681 C ILE 580 18.681 65.556 2.800 1.00 14.39 C ATOM 682 O ILE 580 18.956 66.396 3.643 1.00 15.74 O ATOM 683 CB ILE 580 19.969 65.315 0.673 1.00 10.89 C ATOM 684 CG1 ILE 580 21.209 64.682 0.067 1.00 16.10 C ATOM 685 CG2 ILE 580 20.087 66.835 0.663 1.00 13.34 C ATOM 686 CD1 ILE 580 21.457 65.030 −1.408 1.00 15.50 C ATOM 687 N ALA 581 17.432 65.208 2.508 1.00 16.77 N ATOM 688 CA ALA 581 16.294 65.881 3.121 1.00 16.31 C ATOM 689 C ALA 581 16.252 65.723 4.646 1.00 17.47 C ATOM 690 O ALA 581 15.942 66.670 5.350 1.00 18.94 O ATOM 691 CB ALA 581 14.991 65.386 2.487 1.00 12.38 C ATOM 692 N SER 582 16.559 64.539 5.167 1.00 21.47 N ATOM 693 CA SER 582 16.516 64.344 6.617 1.00 22.46 C ATOM 694 C SER 582 17.700 64.984 7.295 1.00 23.44 C ATOM 695 O SER 582 17.673 65.229 8.497 1.00 29.23 O ATOM 696 CB SER 582 16.469 62.867 6.974 1.00 21.25 C ATOM 697 OG SER 582 17.659 62.228 6.577 1.00 25.04 O ATOM 698 N GLN 583 18.728 65.293 6.522 1.00 22.77 N ATOM 699 CA GLN 583 19.919 65.910 7.077 1.00 23.36 C ATOM 700 C GLN 583 19.874 67.425 6.981 1.00 21.29 C ATOM 701 O GLN 583 20.812 68.117 7.416 1.00 20.02 O ATOM 702 CB GLN 583 21.177 65.369 6.398 1.00 30.71 C ATOM 703 CG GLN 583 21.524 63.939 6.797 1.00 42.85 C ATOM 704 CD GLN 583 22.843 63.455 6.192 1.00 55.80 C ATOM 705 OE1 GLN 583 23.589 62.715 6.839 1.00 62.99 O ATOM 706 NE2 GLN 583 23.141 63.873 4.950 1.00 59.38 N ATOM 707 N VAL 584 18.804 67.940 6.389 1.00 15.51 N ATOM 708 CA VAL 584 18.622 69.371 6.255 1.00 21.88 C ATOM 709 C VAL 584 18.891 70.018 7.615 1.00 26.42 C ATOM 710 O VAL 584 18.367 69.585 8.643 1.00 26.77 O ATOM 711 CB VAL 584 17.194 69.706 5.768 1.00 25.45 C ATOM 712 CG1 VAL 584 16.944 71.199 5.861 1.00 30.17 C ATOM 713 CG2 VAL 584 17.013 69.269 4.333 1.00 27.33 C ATOM 714 N LYS 585 19.690 71.073 7.597 1.00 30.58 N ATOM 715 CA LYS 585 20.108 71.788 8.798 1.00 33.89 C ATOM 716 C LYS 585 19.091 72.734 9.417 1.00 32.68 C ATOM 717 O LYS 585 18.570 73.641 8.747 1.00 33.70 O ATOM 718 CB LYS 585 21.398 72.551 8.497 1.00 41.79 C ATOM 719 CG LYS 585 22.643 71.977 9.165 1.00 52.09 C ATOM 720 CD LYS 585 22.693 72.340 10.657 1.00 63.92 C ATOM 721 CE LYS 585 22.857 73.859 10.873 1.00 70.35 C ATOM 722 NZ LYS 585 22.735 74.271 12.316 1.00 74.91 N ATOM 723 N TYR 586 18.859 72.557 10.718 1.00 30.31 N ATOM 724 CA TYR 586 17.928 73.416 11.454 1.00 29.54 C ATOM 725 C TYR 586 18.583 74.776 11.564 1.00 29.75 C ATOM 726 O TYR 586 19.696 74.885 12.079 1.00 30.67 O ATOM 727 CB TYR 586 17.619 72.856 12.861 1.00 27.95 C ATOM 728 CG TYR 586 16.772 73.793 13.721 1.00 26.56 C ATOM 729 CD1 TYR 586 15.389 73.906 13.522 1.00 24.59 C ATOM 730 CD2 TYR 586 17.361 74.604 14.699 1.00 21.69 C ATOM 731 CE1 TYR 586 14.616 74.806 14.265 1.00 20.84 C ATOM 732 CE2 TYR 586 16.590 75.508 15.457 1.00 17.53 C ATOM 733 CZ TYR 586 15.222 75.601 15.233 1.00 23.50 C ATOM 734 OH TYR 586 14.461 76.482 15.978 1.00 21.84 O ATOM 735 N ALA 587 17.932 75.788 10.994 1.00 28.03 N ATOM 736 CA ALA 587 18.433 77.150 11.016 1.00 27.73 C ATOM 737 C ALA 587 17.945 77.932 12.220 1.00 27.05 C ATOM 738 O ALA 587 18.735 78.601 12.870 1.00 26.39 O ATOM 739 CB ALA 587 18.056 77.882 9.719 1.00 29.03 C ATOM 740 N GLY 588 16.643 77.880 12.490 1.00 25.16 N ATOM 741 CA GLY 588 16.093 78.606 13.619 1.00 24.35 C ATOM 742 C GLY 588 16.224 80.115 13.484 1.00 26.00 C ATOM 743 O GLY 588 16.582 80.806 14.429 1.00 27.67 O ATOM 744 N SER 589 15.891 80.626 12.309 1.00 25.60 N ATOM 745 CA SER 589 15.985 82.044 12.009 1.00 25.91 C ATOM 746 C SER 589 14.916 82.905 12.679 1.00 27.91 C ATOM 747 O SER 589 13.849 82.426 13.050 1.00 23.56 O ATOM 748 CB SER 589 15.879 82.228 10.495 1.00 26.02 C ATOM 749 OG SER 589 16.935 83.028 9.992 1.00 41.28 O ATOM 750 N GLN 590 15.212 84.193 12.811 1.00 32.43 N ATOM 751 CA GLN 590 14.256 85.130 13.373 1.00 37.48 C ATOM 752 C GLN 590 13.253 85.476 12.296 1.00 35.55 C ATOM 753 O GLN 590 12.160 85.937 12.588 1.00 38.88 O ATOM 754 CB GLN 590 14.955 86.415 13.821 1.00 39.80 C ATOM 755 CG GLN 590 15.288 86.435 15.295 1.00 56.15 C ATOM 756 CD GLN 590 16.155 87.622 15.686 1.00 65.66 C ATOM 757 OE1 GLN 590 15.649 88.723 15.932 1.00 71.38 O ATOM 758 NE2 GLN 590 17.473 87.403 15.742 1.00 67.58 N ATOM 759 N VAL 591 13.647 85.246 11.047 1.00 34.29 N ATOM 760 CA VAL 591 12.832 85.570 9.880 1.00 30.91 C ATOM 761 C VAL 591 12.827 84.471 8.809 1.00 26.09 C ATOM 762 O VAL 591 13.885 84.022 8.392 1.00 23.24 O ATOM 763 CB VAL 591 13.359 86.907 9.243 1.00 30.16 C ATOM 764 CG1 VAL 591 13.067 86.983 7.758 1.00 33.78 C ATOM 765 CG2 VAL 591 12.750 88.102 9.949 1.00 29.76 C ATOM 766 N ALA 592 11.636 84.000 8.437 1.00 24.70 N ATOM 767 CA ALA 592 11.462 83.007 7.372 1.00 20.42 C ATOM 768 C ALA 592 10.529 83.712 6.375 1.00 23.74 C ATOM 769 O ALA 592 9.320 83.852 6.629 1.00 19.55 O ATOM 770 CB ALA 592 10.829 81.750 7.893 1.00 14.37 C ATOM 771 N SER 593 11.104 84.209 5.277 1.00 22.94 N ATOM 772 CA SER 593 10.349 84.951 4.270 1.00 23.81 C ATOM 773 C SER 593 9.677 84.093 3.224 1.00 22.86 C ATOM 774 O SER 593 10.336 83.470 2.401 1.00 22.82 O ATOM 775 CB SER 593 11.232 85.974 3.569 1.00 23.57 C ATOM 776 OG SER 593 10.496 86.636 2.556 1.00 14.43 O ATOM 777 N THR 594 8.354 84.150 3.212 1.00 22.76 N ATOM 778 CA THR 594 7.568 83.380 2.263 1.00 22.25 C ATOM 779 C THR 594 7.608 84.069 0.898 1.00 21.75 C ATOM 780 O THR 594 7.593 83.411 −0.143 1.00 21.45 O ATOM 781 CB THR 594 6.122 83.217 2.768 1.00 19.99 C ATOM 782 OG1 THR 594 5.593 84.505 3.106 1.00 18.50 O ATOM 783 CG2 THR 594 6.110 82.375 4.021 1.00 15.10 C ATOM 784 N SER 595 7.663 85.400 0.901 1.00 21.20 N ATOM 785 CA SER 595 7.738 86.164 −0.334 1.00 18.60 C ATOM 786 C SER 595 9.032 85.850 −1.040 1.00 19.69 C ATOM 787 O SER 595 9.044 85.513 −2.228 1.00 22.57 O ATOM 788 CB SER 595 7.661 87.653 −0.037 1.00 11.10 C ATOM 789 OG SER 595 6.344 87.985 0.326 1.00 19.37 O ATOM 790 N GLU 596 10.122 85.922 −0.294 1.00 21.05 N ATOM 791 CA GLU 596 11.418 85.638 −0.863 1.00 23.66 C ATOM 792 C GLU 596 11.591 84.184 −1.299 1.00 22.27 C ATOM 793 O GLU 596 12.302 83.905 −2.260 1.00 24.61 O ATOM 794 CB GLU 596 12.543 86.151 0.043 1.00 30.29 C ATOM 795 CG GLU 596 13.058 87.575 −0.366 1.00 39.81 C ATOM 796 CD GLU 596 11.948 88.663 −0.449 1.00 45.46 C ATOM 797 OE1 GLU 596 11.391 89.067 0.602 1.00 52.14 O ATOM 798 OE2 GLU 596 11.648 89.142 −1.565 1.00 47.05 O ATOM 799 N VAL 597 10.886 83.257 −0.661 1.00 19.54 N ATOM 800 CA VAL 597 10.993 81.869 −1.079 1.00 14.34 C ATOM 801 C VAL 597 10.056 81.566 −2.257 1.00 14.34 C ATOM 802 O VAL 597 10.322 80.677 −3.047 1.00 15.50 O ATOM 803 CB VAL 597 10.768 80.893 0.077 1.00 10.95 C ATOM 804 CG1 VAL 597 9.297 80.603 0.266 1.00 9.01 C ATOM 805 CG2 VAL 597 11.555 79.624 −0.172 1.00 10.66 C ATOM 806 N LEU 598 8.951 82.287 −2.372 1.00 12.70 N ATOM 807 CA LEU 598 8.067 82.083 −3.502 1.00 12.15 C ATOM 808 C LEU 598 8.749 82.749 −4.699 1.00 17.31 C ATOM 809 O LEU 598 8.579 82.324 −5.840 1.00 18.32 O ATOM 810 CB LEU 598 6.700 82.699 −3.240 1.00 8.81 C ATOM 811 CG LEU 598 5.728 81.891 −2.363 1.00 9.60 C ATOM 812 CD1 LEU 598 4.467 82.716 −2.154 1.00 9.06 C ATOM 813 CD2 LEU 598 5.370 80.522 −2.975 1.00 7.06 C ATOM 814 N LYS 599 9.552 83.777 −4.432 1.00 18.09 N ATOM 815 CA LYS 599 10.288 84.442 −5.492 1.00 20.39 C ATOM 816 C LYS 599 11.328 83.475 −6.055 1.00 20.58 C ATOM 817 O LYS 599 11.552 83.432 −7.260 1.00 23.57 O ATOM 818 CB LYS 599 11.001 85.693 −4.982 1.00 21.30 C ATOM 819 CG LYS 599 11.836 86.366 −6.063 1.00 20.62 C ATOM 820 CD LYS 599 12.331 87.732 −5.633 1.00 28.26 C ATOM 821 CE LYS 599 13.674 87.677 −4.914 1.00 28.15 C ATOM 822 NZ LYS 599 14.463 88.927 −5.268 1.00 36.90 N ATOM 823 N TYR 600 11.972 82.712 −5.184 1.00 15.33 N ATOM 824 CA TYR 600 12.975 81.739 −5.605 1.00 14.72 C ATOM 825 C TYR 600 12.380 80.614 −6.470 1.00 15.26 C ATOM 826 O TYR 600 13.031 80.110 −7.394 1.00 14.23 O ATOM 827 CB TYR 600 13.659 81.159 −4.369 1.00 10.22 C ATOM 828 CG TYR 600 14.763 80.168 −4.658 1.00 13.36 C ATOM 829 CD1 TYR 600 16.091 80.594 −4.820 1.00 9.42 C ATOM 830 CD2 TYR 600 14.488 78.791 −4.745 1.00 9.40 C ATOM 831 CE1 TYR 600 17.123 79.670 −5.066 1.00 8.00 C ATOM 832 CE2 TYR 600 15.502 77.874 −4.987 1.00 11.12 C ATOM 833 CZ TYR 600 16.812 78.320 −5.154 1.00 13.46 C ATOM 834 OH TYR 600 17.796 77.412 −5.477 1.00 18.52 O ATOM 835 N THR 601 11.144 80.228 −6.167 1.00 15.74 N ATOM 836 CA THR 601 10.449 79.163 −6.887 1.00 16.26 C ATOM 837 C THR 601 10.062 79.658 −8.291 1.00 18.06 C ATOM 838 O THR 601 10.189 78.942 −9.297 1.00 20.51 O ATOM 839 CB THR 601 9.184 78.724 −6.074 1.00 13.62 C ATOM 840 OG1 THR 601 9.583 78.361 −4.745 1.00 14.88 O ATOM 841 CG2 THR 601 8.493 77.517 −6.690 1.00 8.81 C ATOM 842 N LEU 602 9.660 80.914 −8.358 1.00 18.80 N ATOM 843 CA LEU 602 9.239 81.517 −9.594 1.00 12.28 C ATOM 844 C LEU 602 10.394 81.808 −10.528 1.00 12.80 C ATOM 845 O LEU 602 10.365 81.445 −11.686 1.00 15.88 O ATOM 846 CB LEU 602 8.497 82.800 −9.259 1.00 7.41 C ATOM 847 CG LEU 602 7.971 83.645 −10.410 1.00 4.81 C ATOM 848 CD1 LEU 602 7.137 82.788 −11.305 1.00 11.16 C ATOM 849 CD2 LEU 602 7.163 84.783 −9.881 1.00 3.73 C ATOM 850 N PHE 603 11.409 82.496 −10.031 1.00 17.52 N ATOM 851 CA PHE 603 12.537 82.883 −10.862 1.00 18.59 C ATOM 852 C PHE 603 13.743 81.966 −10.844 1.00 21.46 C ATOM 853 O PHE 603 14.577 82.022 −11.746 1.00 21.41 O ATOM 854 CB PHE 603 12.954 84.322 −10.533 1.00 13.06 C ATOM 855 CG PHE 603 11.860 85.333 −10.758 1.00 13.57 C ATOM 856 CD1 PHE 603 11.257 85.469 −12.006 1.00 13.30 C ATOM 857 CD2 PHE 603 11.422 86.138 −9.722 1.00 11.61 C ATOM 858 CE1 PHE 603 10.232 86.385 −12.212 1.00 8.39 C ATOM 859 CE2 PHE 603 10.403 87.054 −9.916 1.00 14.44 C ATOM 860 CZ PHE 603 9.804 87.180 −11.160 1.00 14.78 C ATOM 861 N GLN 604 13.828 81.087 −9.856 1.00 24.28 N ATOM 862 CA GLN 604 14.983 80.205 −9.787 1.00 27.12 C ATOM 863 C GLN 604 14.687 78.739 −10.058 1.00 24.50 C ATOM 864 O GLN 604 15.512 78.040 −10.605 1.00 24.04 O ATOM 865 CB GLN 604 15.689 80.368 −8.440 1.00 31.32 C ATOM 866 CG GLN 604 16.522 81.621 −8.287 1.00 35.80 C ATOM 867 CD GLN 604 17.861 81.469 −8.960 1.00 45.50 C ATOM 868 OE1 GLN 604 18.784 80.859 −8.418 1.00 49.26 O ATOM 869 NE2 GLN 604 17.973 81.999 −10.166 1.00 54.22 N ATOM 870 N ILE 605 13.521 78.260 −9.660 1.00 23.49 N ATOM 871 CA ILE 605 13.196 76.859 −9.881 1.00 22.06 C ATOM 872 C ILE 605 12.418 76.627 −11.163 1.00 25.79 C ATOM 873 O ILE 605 12.635 75.633 −11.861 1.00 28.80 O ATOM 874 CB ILE 605 12.425 76.243 −8.678 1.00 18.12 C ATOM 875 CG1 ILE 605 13.356 76.119 −7.470 1.00 16.09 C ATOM 876 CG2 ILE 605 11.862 74.874 −9.035 1.00 15.46 C ATOM 877 CD1 ILE 605 12.661 75.604 −6.225 1.00 12.12 C ATOM 878 N PHE 606 11.444 77.484 −11.431 1.00 21.94 N ATOM 879 CA PHE 606 10.670 77.333 −12.645 1.00 22.03 C ATOM 880 C PHE 606 10.903 78.556 −13.533 1.00 22.92 C ATOM 881 O PHE 606 9.965 79.130 −14.083 1.00 19.90 O ATOM 882 CB PHE 606 9.188 77.149 −12.310 1.00 19.38 C ATOM 883 CG PHE 606 8.899 75.943 −11.442 1.00 24.03 C ATOM 884 CD1 PHE 606 8.941 74.656 −11.969 1.00 21.11 C ATOM 885 CD2 PHE 606 8.498 76.107 −10.108 1.00 23.28 C ATOM 886 CE1 PHE 606 8.582 73.560 −11.178 1.00 22.09 C ATOM 887 CE2 PHE 606 8.141 75.025 −9.319 1.00 19.77 C ATOM 888 CZ PHE 606 8.175 73.749 −9.850 1.00 22.92 C ATOM 889 N SER 607 12.165 78.971 −13.641 1.00 27.16 N ATOM 890 CA SER 607 12.505 80.127 −14.456 1.00 34.01 C ATOM 891 C SER 607 11.927 79.863 −15.832 1.00 41.74 C ATOM 892 O SER 607 11.276 80.737 −16.421 1.00 49.40 O ATOM 893 CB SER 607 13.997 80.342 −14.543 1.00 29.88 C ATOM 894 OG SER 607 14.209 81.653 −15.023 1.00 31.36 O ATOM 895 N LYS 608 12.279 78.717 −16.404 1.00 43.35 N ATOM 896 CA LYS 608 11.647 78.316 −17.648 1.00 46.17 C ATOM 897 C LYS 608 11.480 76.817 −17.716 1.00 42.64 C ATOM 898 O LYS 608 12.365 76.029 −17.390 1.00 40.78 O ATOM 899 CB LYS 608 12.195 78.939 −18.935 1.00 54.48 C ATOM 900 CG LYS 608 11.035 79.221 −19.979 1.00 55.51 C ATOM 901 CD LYS 608 9.997 80.303 −19.504 1.00 54.84 C ATOM 902 CE LYS 608 8.628 79.732 −19.088 1.00 53.13 C ATOM 903 NZ LYS 608 7.777 79.171 −20.196 1.00 50.29 N ATOM 904 N ILE 609 10.244 76.475 −18.018 1.00 38.42 N ATOM 905 CA ILE 609 9.753 75.134 −18.077 1.00 32.87 C ATOM 906 C ILE 609 10.437 74.185 −19.038 1.00 30.65 C ATOM 907 O ILE 609 10.108 74.152 −20.208 1.00 34.42 O ATOM 908 CB ILE 609 8.235 75.198 −18.302 1.00 31.16 C ATOM 909 CG1 ILE 609 7.626 76.217 −17.324 1.00 30.77 C ATOM 910 CG2 ILE 609 7.595 73.836 −18.148 1.00 32.93 C ATOM 911 CD1 ILE 609 8.177 76.152 −15.893 1.00 27.01 C ATOM 912 N ASP 610 11.382 73.407 −18.512 1.00 29.30 N ATOM 913 CA ASP 610 12.115 72.397 −19.278 1.00 26.65 C ATOM 914 C ASP 610 11.296 71.090 −19.348 1.00 24.52 C ATOM 915 O ASP 610 11.667 70.160 −20.061 1.00 22.38 O ATOM 916 CB ASP 610 13.472 72.100 −18.610 1.00 31.38 C ATOM 917 CG ASP 610 13.326 71.445 −17.208 1.00 40.16 C ATOM 918 OD1 ASP 610 12.777 72.111 −16.296 1.00 44.05 O ATOM 919 OD2 ASP 610 13.781 70.280 −17.006 1.00 37.91 O ATOM 920 N ARG 611 10.207 71.023 −18.586 1.00 20.53 N ATOM 921 CA ARG 611 9.338 69.847 −18.523 1.00 17.10 C ATOM 922 C ARG 611 7.882 70.318 −18.565 1.00 18.14 C ATOM 923 O ARG 611 7.147 70.151 −17.579 1.00 17.77 O ATOM 924 CB ARG 611 9.555 69.117 −17.188 1.00 15.35 C ATOM 925 CG ARG 611 10.090 67.715 −17.279 1.00 19.43 C ATOM 926 CD ARG 611 9.053 66.659 −16.878 1.00 17.95 C ATOM 927 NE ARG 611 9.225 66.220 −15.507 1.00 15.53 N ATOM 928 CZ ARG 611 8.787 65.070 −15.009 1.00 15.73 C ATOM 929 NH1 ARG 611 8.143 64.187 −15.746 1.00 14.15 N ATOM 930 NH2 ARG 611 8.950 64.830 −13.726 1.00 20.70 N ATOM 931 N PRO 612 7.410 70.799 −19.734 1.00 18.33 N ATOM 932 CA PRO 612 6.023 71.278 −19.846 1.00 18.14 C ATOM 933 C PRO 612 4.985 70.176 −19.746 1.00 16.54 C ATOM 934 O PRO 612 3.800 70.458 −19.694 1.00 18.83 O ATOM 935 CB PRO 612 6.003 71.928 −21.226 1.00 20.27 C ATOM 936 CG PRO 612 6.909 70.998 −22.015 1.00 21.20 C ATOM 937 CD PRO 612 8.066 70.757 −21.054 1.00 19.55 C ATOM 938 N GLU 613 5.439 68.926 −19.694 1.00 17.56 N ATOM 939 CA GLU 613 4.539 67.780 −19.590 1.00 18.04 C ATOM 940 C GLU 613 4.265 67.341 −18.137 1.00 20.09 C ATOM 941 O GLU 613 3.525 66.376 −17.895 1.00 19.26 O ATOM 942 CB GLU 613 5.088 66.593 −20.380 1.00 17.32 C ATOM 943 CG GLU 613 6.320 65.939 −19.764 1.00 16.50 C ATOM 944 CD GLU 613 7.622 66.575 −20.203 1.00 17.75 C ATOM 945 OE1 GLU 613 7.609 67.682 −20.775 1.00 21.13 O ATOM 946 OE2 GLU 613 8.680 65.949 −19.979 1.00 18.68 O ATOM 947 N ALA 614 4.896 67.997 −17.172 1.00 17.80 N ATOM 948 CA ALA 614 4.645 67.643 −15.782 1.00 17.35 C ATOM 949 C ALA 614 3.998 68.805 −15.068 1.00 17.82 C ATOM 950 O ALA 614 4.245 69.962 −15.422 1.00 18.03 O ATOM 951 CB ALA 614 5.929 67.302 −15.103 1.00 14.29 C ATOM 952 N SER 615 3.143 68.502 −14.088 1.00 14.90 N ATOM 953 CA SER 615 2.532 69.553 −13.297 1.00 13.08 C ATOM 954 C SER 615 3.622 70.036 −12.347 1.00 14.27 C ATOM 955 O SER 615 4.577 69.304 −12.056 1.00 17.25 O ATOM 956 CB SER 615 1.362 69.029 −12.491 1.00 10.01 C ATOM 957 OG SER 615 0.379 68.502 −13.348 1.00 13.32 O ATOM 958 N ARG 616 3.505 71.290 −11.929 1.00 13.79 N ATOM 959 CA ARG 616 4.441 71.923 −11.021 1.00 10.29 C ATOM 960 C ARG 616 3.696 72.218 −9.722 1.00 8.30 C ATOM 961 O ARG 616 2.638 72.841 −9.721 1.00 8.31 O ATOM 962 CB ARG 616 4.966 73.188 −11.671 1.00 10.60 C ATOM 963 CG ARG 616 5.580 72.883 −13.020 1.00 11.29 C ATOM 964 CD ARG 616 5.648 74.101 −13.902 1.00 18.79 C ATOM 965 NE ARG 616 5.380 73.740 −15.286 1.00 25.65 N ATOM 966 CZ ARG 616 4.217 73.955 −15.877 1.00 28.89 C ATOM 967 NH1 ARG 616 3.238 74.543 −15.223 1.00 36.05 N ATOM 968 NH2 ARG 616 3.977 73.463 −17.075 1.00 37.22 N ATOM 969 N ILE 617 4.187 71.673 −8.616 1.00 10.46 N ATOM 970 CA ILE 617 3.532 71.893 −7.341 1.00 8.09 C ATOM 971 C ILE 617 4.549 72.311 −6.289 1.00 9.44 C ATOM 972 O ILE 617 5.625 71.717 −6.176 1.00 13.23 O ATOM 973 CB ILE 617 2.790 70.616 −6.898 1.00 13.19 C ATOM 974 CG1 ILE 617 1.747 70.212 −7.951 1.00 12.54 C ATOM 975 CG2 ILE 617 2.102 70.832 −5.549 1.00 17.10 C ATOM 976 CD1 ILE 617 0.818 69.049 −7.489 1.00 9.40 C ATOM 977 N ALA 618 4.241 73.396 −5.587 1.00 11.26 N ATOM 978 CA ALA 618 5.092 73.932 −4.523 1.00 9.88 C ATOM 979 C ALA 618 4.319 73.708 −3.221 1.00 13.13 C ATOM 980 O ALA 618 3.164 74.135 −3.084 1.00 13.87 O ATOM 981 CB ALA 618 5.332 75.403 −4.747 1.00 6.93 C ATOM 982 N LEU 619 4.920 72.964 −2.304 1.00 11.48 N ATOM 983 CA LEU 619 4.286 72.655 −1.029 1.00 14.03 C ATOM 984 C LEU 619 4.900 73.622 −0.022 1.00 12.75 C ATOM 985 O LEU 619 6.035 73.447 0.395 1.00 9.90 O ATOM 986 CB LEU 619 4.604 71.206 −0.675 1.00 14.36 C ATOM 987 CG LEU 619 3.800 70.416 0.353 1.00 19.68 C ATOM 988 CD1 LEU 619 2.311 70.465 0.078 1.00 19.00 C ATOM 989 CD2 LEU 619 4.319 68.989 0.289 1.00 14.02 C ATOM 990 N LEU 620 4.186 74.700 0.281 1.00 11.45 N ATOM 991 CA LEU 620 4.706 75.711 1.201 1.00 14.00 C ATOM 992 C LEU 620 4.397 75.384 2.663 1.00 13.84 C ATOM 993 O LEU 620 3.271 75.581 3.123 1.00 10.40 O ATOM 994 CB LEU 620 4.156 77.095 0.823 1.00 11.10 C ATOM 995 CG LEU 620 4.779 78.300 1.525 1.00 10.11 C ATOM 996 CD1 LEU 620 6.276 78.366 1.250 1.00 10.19 C ATOM 997 CD2 LEU 620 4.089 79.568 1.058 1.00 11.71 C ATOM 998 N LEU 621 5.397 74.880 3.380 1.00 12.67 N ATOM 999 CA LEU 621 5.226 74.528 4.785 1.00 15.42 C ATOM 1000 C LEU 621 5.536 75.770 5.639 1.00 15.14 C ATOM 1001 O LEU 621 6.697 76.117 5.882 1.00 17.05 O ATOM 1002 CB LEU 621 6.117 73.327 5.100 1.00 11.95 C ATOM 1003 CG LEU 621 5.761 72.185 4.115 1.00 11.75 C ATOM 1004 CD1 LEU 621 6.957 71.338 3.727 1.00 7.16 C ATOM 1005 CD2 LEU 621 4.678 71.351 4.702 1.00 7.13 C ATOM 1006 N MET 622 4.467 76.390 6.131 1.00 13.49 N ATOM 1007 CA MET 622 4.538 77.624 6.899 1.00 17.11 C ATOM 1008 C MET 622 4.273 77.505 8.396 1.00 17.35 C ATOM 1009 O MET 622 3.211 77.062 8.803 1.00 18.55 O ATOM 1010 CB MET 622 3.499 78.593 6.341 1.00 19.32 C ATOM 1011 CG MET 622 3.634 78.898 4.878 1.00 19.64 C ATOM 1012 SD MET 622 2.174 79.715 4.255 1.00 22.75 S ATOM 1013 CE MET 622 2.302 81.305 4.988 1.00 20.32 C ATOM 1014 N ALA 623 5.199 78.003 9.204 1.00 16.59 N ATOM 1015 CA ALA 623 5.057 78.000 10.654 1.00 14.97 C ATOM 1016 C ALA 623 5.115 79.425 11.201 1.00 18.32 C ATOM 1017 O ALA 623 4.952 79.648 12.405 1.00 21.62 O ATOM 1018 CB ALA 623 6.167 77.173 11.279 1.00 15.96 C ATOM 1019 N SER 624 5.300 80.397 10.323 1.00 19.46 N ATOM 1020 CA SER 624 5.433 81.772 10.771 1.00 21.55 C ATOM 1021 C SER 624 4.869 82.812 9.799 1.00 22.83 C ATOM 1022 O SER 624 4.108 82.483 8.855 1.00 18.76 O ATOM 1023 CB SER 624 6.921 82.065 11.026 1.00 18.76 C ATOM 1024 OG SER 624 7.721 81.753 9.896 1.00 22.42 O ATOM 1025 N GLN 625 5.152 84.075 10.122 1.00 22.23 N ATOM 1026 CA GLN 625 4.776 85.204 9.282 1.00 27.39 C ATOM 1027 C GLN 625 5.948 86.161 9.222 1.00 27.09 C ATOM 1028 O GLN 625 6.583 86.445 10.227 1.00 27.21 O ATOM 1029 CB GLN 625 3.498 85.908 9.742 1.00 27.20 C ATOM 1030 CG GLN 625 3.294 86.097 11.206 1.00 33.26 C ATOM 1031 CD GLN 625 1.881 86.577 11.507 1.00 32.60 C ATOM 1032 OE1 GLN 625 1.168 85.979 12.305 1.00 41.09 O ATOM 1033 NE2 GLN 625 1.471 87.647 10.854 1.00 32.88 N ATOM 1034 N GLU 626 6.326 86.519 8.004 1.00 28.00 N ATOM 1035 CA GLU 626 7.434 87.437 7.771 1.00 28.38 C ATOM 1036 C GLU 626 7.013 88.885 8.071 1.00 29.02 C ATOM 1037 O GLU 626 5.813 89.221 8.041 1.00 25.62 O ATOM 1038 CB GLU 626 7.852 87.355 6.304 1.00 25.11 C ATOM 1039 CG GLU 626 6.807 87.963 5.378 1.00 23.18 C ATOM 1040 CD GLU 626 7.062 87.693 3.915 1.00 24.81 C ATOM 1041 OE1 GLU 626 8.171 87.208 3.580 1.00 20.13 O ATOM 1042 OE2 GLU 626 6.129 87.946 3.116 1.00 17.26 O ATOM 1043 N PRO 627 7.995 89.762 8.367 1.00 29.70 N ATOM 1044 CA PRO 627 7.708 91.173 8.653 1.00 31.82 C ATOM 1045 C PRO 627 7.050 91.835 7.441 1.00 33.52 C ATOM 1046 O PRO 627 7.505 91.662 6.310 1.00 33.48 O ATOM 1047 CB PRO 627 9.096 91.759 8.947 1.00 28.98 C ATOM 1048 CG PRO 627 10.037 90.803 8.297 1.00 31.86 C ATOM 1049 CD PRO 627 9.420 89.477 8.587 1.00 29.78 C ATOM 1050 N GLN 628 6.000 92.609 7.702 1.00 36.73 N ATOM 1051 CA GLN 628 5.208 93.302 6.679 1.00 38.04 C ATOM 1052 C GLN 628 6.026 93.896 5.542 1.00 35.21 C ATOM 1053 O GLN 628 5.629 93.832 4.375 1.00 34.60 O ATOM 1054 CB GLN 628 4.353 94.406 7.332 1.00 44.33 C ATOM 1055 CG GLN 628 2.922 94.535 6.808 1.00 52.77 C ATOM 1056 CD GLN 628 1.885 94.095 7.834 1.00 60.03 C ATOM 1057 OE1 GLN 628 1.070 94.899 8.302 1.00 61.34 O ATOM 1058 NE2 GLN 628 1.923 92.814 8.206 1.00 63.65 N ATOM 1059 N ARG 629 7.180 94.448 5.884 1.00 32.79 N ATOM 1060 CA ARG 629 8.051 95.067 4.898 1.00 34.31 C ATOM 1061 C ARG 629 8.588 94.118 3.822 1.00 30.81 C ATOM 1062 O ARG 629 8.919 94.556 2.723 1.00 36.74 O ATOM 1063 CB ARG 629 9.209 95.784 5.592 1.00 38.79 C ATOM 1064 CG ARG 629 10.042 94.861 6.431 1.00 47.03 C ATOM 1065 CD ARG 629 11.399 95.422 6.687 1.00 49.94 C ATOM 1066 NE ARG 629 12.324 94.336 6.978 1.00 58.97 N ATOM 1067 CZ ARG 629 13.603 94.339 6.633 1.00 65.16 C ATOM 1068 NH1 ARG 629 14.109 95.379 5.985 1.00 70.46 N ATOM 1069 NH2 ARG 629 14.378 93.314 6.952 1.00 68.49 N ATOM 1070 N MET 630 8.655 92.828 4.110 1.00 25.21 N ATOM 1071 CA MET 630 9.171 91.879 3.134 1.00 23.72 C ATOM 1072 C MET 630 8.061 91.297 2.268 1.00 24.23 C ATOM 1073 O MET 630 8.346 90.631 1.270 1.00 25.88 O ATOM 1074 CB MET 630 9.905 90.735 3.828 1.00 25.55 C ATOM 1075 CG MET 630 11.078 91.153 4.687 1.00 28.67 C ATOM 1076 SD MET 630 11.844 89.733 5.470 1.00 30.08 S ATOM 1077 CE MET 630 13.015 89.228 4.233 1.00 33.64 C ATOM 1078 N SER 631 6.811 91.617 2.606 1.00 23.98 N ATOM 1079 CA SER 631 5.634 91.098 1.906 1.00 24.27 C ATOM 1080 C SER 631 5.076 91.919 0.751 1.00 26.26 C ATOM 1081 O SER 631 4.010 91.584 0.210 1.00 21.34 O ATOM 1082 CB SER 631 4.504 90.879 2.911 1.00 22.54 C ATOM 1083 OG SER 631 4.923 90.050 3.983 1.00 24.17 O ATOM 1084 N ARG 632 5.797 92.967 0.357 1.00 29.47 N ATOM 1085 CA ARG 632 5.348 93.865 −0.711 1.00 27.92 C ATOM 1086 C ARG 632 4.990 93.255 −2.075 1.00 24.00 C ATOM 1087 O ARG 632 4.013 93.653 −2.698 1.00 19.20 O ATOM 1088 CB ARG 632 6.350 95.003 −0.869 1.00 32.62 C ATOM 1089 CG ARG 632 6.093 96.167 0.064 1.00 38.66 C ATOM 1090 CD ARG 632 7.198 97.197 −0.041 1.00 48.62 C ATOM 1091 NE ARG 632 8.267 96.958 0.932 1.00 58.66 N ATOM 1092 CZ ARG 632 9.535 97.343 0.786 1.00 61.73 C ATOM 1093 NH1 ARG 632 9.927 97.984 −0.317 1.00 62.36 N ATOM 1094 NH2 ARG 632 10.396 97.139 1.779 1.00 60.51 N ATOM 1095 N ASN 633 5.754 92.267 −2.520 1.00 22.34 N ATOM 1096 CA ASN 633 5.501 91.630 −3.812 1.00 23.39 C ATOM 1097 C ASN 633 4.823 90.274 −3.673 1.00 22.81 C ATOM 1098 O ASN 633 4.613 89.592 −4.665 1.00 20.30 O ATOM 1099 CB ASN 633 6.815 91.430 −4.583 1.00 19.08 C ATOM 1100 CG ASN 633 7.352 92.708 −5.169 1.00 17.97 C ATOM 1101 OD1 ASN 633 6.663 93.724 −5.197 1.00 12.49 O ATOM 1102 ND2 ASN 633 8.602 92.667 −5.644 1.00 17.03 N ATOM 1103 N PHE 634 4.494 89.889 −2.445 1.00 22.28 N ATOM 1104 CA PHE 634 3.878 88.603 −2.182 1.00 19.74 C ATOM 1105 C PHE 634 2.821 88.172 −3.190 1.00 18.80 C ATOM 1106 O PHE 634 2.994 87.142 −3.849 1.00 19.49 O ATOM 1107 CB PHE 634 3.303 88.536 −0.756 1.00 14.84 C ATOM 1108 CG PHE 634 2.736 87.184 −0.414 1.00 11.94 C ATOM 1109 CD1 PHE 634 3.583 86.149 −0.048 1.00 4.10 C ATOM 1110 CD2 PHE 634 1.373 86.920 −0.576 1.00 11.23 C ATOM 1111 CE1 PHE 634 3.095 84.880 0.136 1.00 5.83 C ATOM 1112 CE2 PHE 634 0.858 85.645 −0.393 1.00 10.88 C ATOM 1113 CZ PHE 634 1.719 84.617 −0.040 1.00 9.89 C ATOM 1114 N VAL 635 1.751 88.942 −3.365 1.00 18.69 N ATOM 1115 CA VAL 635 0.707 88.514 −4.314 1.00 21.28 C ATOM 1116 C VAL 635 1.214 88.374 −5.751 1.00 21.28 C ATOM 1117 O VAL 635 0.803 87.478 −6.480 1.00 22.82 O ATOM 1118 CB VAL 635 −0.500 89.440 −4.280 1.00 20.51 C ATOM 1119 CG1 VAL 635 −1.510 89.022 −5.316 1.00 25.23 C ATOM 1120 CG2 VAL 635 −1.129 89.383 −2.922 1.00 23.14 C ATOM 1121 N ARG 636 2.160 89.227 −6.111 1.00 18.25 N ATOM 1122 CA ARG 636 2.778 89.239 −7.420 1.00 13.92 C ATOM 1123 C ARG 636 3.522 87.936 −7.677 1.00 15.53 C ATOM 1124 O ARG 636 3.419 87.366 −8.760 1.00 19.16 O ATOM 1125 CB ARG 636 3.719 90.446 −7.480 1.00 13.52 C ATOM 1126 CG ARG 636 2.923 91.760 −7.492 1.00 21.06 C ATOM 1127 CD ARG 636 3.742 92.986 −7.115 1.00 31.89 C ATOM 1128 NE ARG 636 3.059 94.231 −7.497 1.00 34.04 N ATOM 1129 CZ ARG 636 2.973 95.330 −6.747 1.00 33.28 C ATOM 1130 NH1 ARG 636 3.523 95.373 −5.545 1.00 32.30 N ATOM 1131 NH2 ARG 636 2.396 96.421 −7.233 1.00 35.94 N ATOM 1132 N TYR 637 4.249 87.439 −6.683 1.00 12.24 N ATOM 1133 CA TYR 637 4.969 86.187 −6.837 1.00 13.29 C ATOM 1134 C TYR 637 3.984 85.046 −6.970 1.00 15.41 C ATOM 1135 O TYR 637 4.176 84.141 −7.789 1.00 17.67 O ATOM 1136 CB TYR 637 5.912 85.942 −5.654 1.00 16.02 C ATOM 1137 CG TYR 637 7.003 86.987 −5.572 1.00 17.66 C ATOM 1138 CD1 TYR 637 7.556 87.512 −6.726 1.00 23.47 C ATOM 1139 CD2 TYR 637 7.438 87.486 −4.356 1.00 23.35 C ATOM 1140 CE1 TYR 637 8.507 88.512 −6.684 1.00 24.21 C ATOM 1141 CE2 TYR 637 8.398 88.493 −4.296 1.00 25.27 C ATOM 1142 CZ TYR 637 8.929 89.000 −5.471 1.00 27.11 C ATOM 1143 OH TYR 637 9.897 89.989 −5.446 1.00 27.67 O ATOM 1144 N VAL 638 2.892 85.114 −6.212 1.00 17.90 N ATOM 1145 CA VAL 638 1.866 84.073 −6.264 1.00 15.71 C ATOM 1146 C VAL 638 1.110 84.107 −7.591 1.00 16.42 C ATOM 1147 O VAL 638 0.730 83.057 −8.121 1.00 13.74 O ATOM 1148 CB VAL 638 0.879 84.189 −5.070 1.00 14.51 C ATOM 1149 CG1 VAL 638 −0.224 83.196 −5.201 1.00 10.64 C ATOM 1150 CG2 VAL 638 1.623 83.907 −3.762 1.00 15.68 C ATOM 1151 N GLN 639 0.858 85.306 −8.122 1.00 17.70 N ATOM 1152 CA GLN 639 0.173 85.418 −9.401 1.00 19.16 C ATOM 1153 C GLN 639 1.132 84.973 −10.494 1.00 20.06 C ATOM 1154 O GLN 639 0.705 84.381 −11.500 1.00 21.31 O ATOM 1155 CB GLN 639 −0.280 86.842 −9.661 1.00 24.29 C ATOM 1156 CG GLN 639 −1.377 87.339 −8.758 1.00 33.20 C ATOM 1157 CD GLN 639 −1.509 88.855 −8.824 1.00 42.75 C ATOM 1158 OE1 GLN 639 −0.534 89.573 −9.095 1.00 47.34 O ATOM 1159 NE2 GLN 639 −2.709 89.353 −8.572 1.00 47.82 N ATOM 1160 N GLY 640 2.428 85.212 −10.272 1.00 19.08 N ATOM 1161 CA GLY 640 3.465 84.789 −11.212 1.00 16.51 C ATOM 1162 C GLY 640 3.515 83.268 −11.342 1.00 16.94 C ATOM 1163 O GLY 640 3.492 82.740 −12.454 1.00 19.48 O ATOM 1164 N LEU 641 3.553 82.551 −10.221 1.00 16.74 N ATOM 1165 CA LEU 641 3.579 81.088 −10.252 1.00 13.10 C ATOM 1166 C LEU 641 2.269 80.555 −10.792 1.00 13.33 C ATOM 1167 O LEU 641 2.206 79.465 −11.355 1.00 18.15 O ATOM 1168 CB LEU 641 3.802 80.530 −8.852 1.00 14.38 C ATOM 1169 CG LEU 641 5.198 80.681 −8.238 1.00 16.65 C ATOM 1170 CD1 LEU 641 5.139 80.462 −6.733 1.00 18.86 C ATOM 1171 CD2 LEU 641 6.206 79.737 −8.886 1.00 6.08 C ATOM 1172 N LYS 642 1.205 81.311 −10.605 1.00 14.11 N ATOM 1173 CA LYS 642 −0.101 80.898 −11.096 1.00 16.06 C ATOM 1174 C LYS 642 −0.150 80.870 −12.623 1.00 16.97 C ATOM 1175 O LYS 642 −0.809 80.019 −13.210 1.00 19.62 O ATOM 1176 CB LYS 642 −1.169 81.847 −10.555 1.00 16.72 C ATOM 1177 CG LYS 642 −2.558 81.585 −11.078 1.00 18.86 C ATOM 1178 CD LYS 642 −3.085 82.761 −11.881 1.00 14.53 C ATOM 1179 CE LYS 642 −4.522 82.498 −12.267 1.00 25.13 C ATOM 1180 NZ LYS 642 −5.053 83.581 −13.128 1.00 33.85 N ATOM 1181 N LYS 643 0.517 81.829 −13.256 1.00 17.23 N ATOM 1182 CA LYS 643 0.554 81.933 −14.714 1.00 15.33 C ATOM 1183 C LYS 643 1.409 80.845 −15.322 1.00 14.03 C ATOM 1184 O LYS 643 1.183 80.439 −16.464 1.00 13.60 O ATOM 1185 CB LYS 643 1.127 83.281 −15.127 1.00 16.90 C ATOM 1186 CG LYS 643 0.301 84.453 −14.692 1.00 23.60 C ATOM 1187 CD LYS 643 0.990 85.751 −15.080 1.00 29.59 C ATOM 1188 CE LYS 643 0.321 86.922 −14.390 1.00 34.30 C ATOM 1189 NZ LYS 643 1.216 88.106 −14.326 1.00 35.59 N ATOM 1190 N LYS 644 2.442 80.431 −14.598 1.00 15.71 N ATOM 1191 CA LYS 644 3.321 79.373 −15.059 1.00 15.56 C ATOM 1192 C LYS 644 2.720 78.041 −14.644 1.00 13.62 C ATOM 1193 O LYS 644 3.351 77.009 −14.825 1.00 16.26 O ATOM 1194 CB LYS 644 4.737 79.517 −14.466 1.00 19.22 C ATOM 1195 CG LYS 644 5.710 80.398 −15.264 1.00 22.27 C ATOM 1196 CD LYS 644 7.123 80.272 −14.690 1.00 24.71 C ATOM 1197 CE LYS 644 8.123 81.299 −15.260 1.00 31.85 C ATOM 1198 NZ LYS 644 9.118 81.871 −14.243 1.00 23.70 N ATOM 1199 N LYS 645 1.502 78.076 −14.098 1.00 10.95 N ATOM 1200 CA LYS 645 0.761 76.876 −13.644 1.00 14.53 C ATOM 1201 C LYS 645 1.425 76.068 −12.527 1.00 15.63 C ATOM 1202 O LYS 645 1.324 74.833 −12.481 1.00 10.70 O ATOM 1203 CB LYS 645 0.397 75.957 −14.803 1.00 14.90 C ATOM 1204 CG LYS 645 −1.087 75.949 −15.174 1.00 16.56 C ATOM 1205 CD LYS 645 −1.866 75.078 −14.228 1.00 19.52 C ATOM 1206 CE LYS 645 −3.242 75.666 −13.949 1.00 28.98 C ATOM 1207 NZ LYS 645 −4.386 74.911 −14.522 1.00 29.12 N ATOM 1208 N VAL 646 2.095 76.784 −11.630 1.00 13.98 N ATOM 1209 CA VAL 646 2.745 76.182 −10.481 1.00 17.17 C ATOM 1210 C VAL 646 1.723 76.303 −9.379 1.00 17.08 C ATOM 1211 O VAL 646 1.333 77.413 −9.028 1.00 15.83 O ATOM 1212 CB VAL 646 4.004 76.952 −10.065 1.00 15.22 C ATOM 1213 CG1 VAL 646 4.628 76.308 −8.805 1.00 14.08 C ATOM 1214 CG2 VAL 646 4.980 76.955 −11.204 1.00 10.59 C ATOM 1215 N ILE 647 1.247 75.160 −8.895 1.00 17.86 N ATOM 1216 CA ILE 647 0.235 75.107 −7.852 1.00 15.74 C ATOM 1217 C ILE 647 0.858 75.303 −6.464 1.00 15.68 C ATOM 1218 O ILE 647 1.785 74.592 −6.086 1.00 17.61 O ATOM 1219 CB ILE 647 −0.514 73.765 −7.947 1.00 14.07 C ATOM 1220 CG1 ILE 647 −1.094 73.600 −9.364 1.00 17.76 C ATOM 1221 CG2 ILE 647 −1.620 73.665 −6.896 1.00 12.37 C ATOM 1222 CD1 ILE 647 −1.913 74.795 −9.865 1.00 11.46 C ATOM 1223 N VAL 648 0.400 76.298 −5.722 1.00 16.62 N ATOM 1224 CA VAL 648 0.969 76.519 −4.387 1.00 16.18 C ATOM 1225 C VAL 648 0.004 75.932 −3.365 1.00 15.96 C ATOM 1226 O VAL 648 −1.158 76.338 −3.284 1.00 14.76 O ATOM 1227 CB VAL 648 1.258 78.035 −4.079 1.00 12.13 C ATOM 1228 CG1 VAL 648 2.023 78.179 −2.775 1.00 9.31 C ATOM 1229 CG2 VAL 648 2.078 78.678 −5.223 1.00 11.58 C ATOM 1230 N ILE 649 0.456 74.906 −2.659 1.00 15.66 N ATOM 1231 CA ILE 649 −0.369 74.276 −1.644 1.00 16.78 C ATOM 1232 C ILE 649 0.222 74.671 −0.306 1.00 17.79 C ATOM 1233 O ILE 649 1.223 74.095 0.124 1.00 18.08 O ATOM 1234 CB ILE 649 −0.376 72.747 −1.769 1.00 14.88 C ATOM 1235 CG1 ILE 649 −0.873 72.352 −3.159 1.00 16.15 C ATOM 1236 CG2 ILE 649 −1.297 72.142 −0.708 1.00 16.62 C ATOM 1237 CD1 ILE 649 −0.925 70.873 −3.418 1.00 17.42 C ATOM 1238 N PRO 650 −0.351 75.709 0.334 1.00 16.08 N ATOM 1239 CA PRO 650 0.076 76.238 1.627 1.00 12.67 C ATOM 1240 C PRO 650 −0.300 75.297 2.775 1.00 15.98 C ATOM 1241 O PRO 650 −1.478 74.921 2.940 1.00 13.96 O ATOM 1242 CB PRO 650 −0.737 77.512 1.764 1.00 9.54 C ATOM 1243 CG PRO 650 −1.475 77.687 0.465 1.00 13.54 C ATOM 1244 CD PRO 650 −1.626 76.326 −0.068 1.00 16.46 C ATOM 1245 N VAL 651 0.693 74.893 3.553 1.00 14.32 N ATOM 1246 CA VAL 651 0.422 74.040 4.697 1.00 14.36 C ATOM 1247 C VAL 651 0.728 74.922 5.911 1.00 12.69 C ATOM 1248 O VAL 651 1.871 75.307 6.129 1.00 15.37 O ATOM 1249 CB VAL 651 1.293 72.782 4.691 1.00 14.81 C ATOM 1250 CG1 VAL 651 0.942 71.918 5.884 1.00 15.50 C ATOM 1251 CG2 VAL 651 1.075 72.004 3.392 1.00 10.07 C ATOM 1252 N GLY 652 −0.307 75.355 6.617 1.00 9.84 N ATOM 1253 CA GLY 652 −0.102 76.201 7.776 1.00 12.11 C ATOM 1254 C GLY 652 0.074 75.324 8.991 1.00 11.33 C ATOM 1255 O GLY 652 −0.778 74.503 9.244 1.00 17.62 O ATOM 1256 N ILE 653 1.150 75.492 9.742 1.00 13.97 N ATOM 1257 CA ILE 653 1.397 74.679 10.931 1.00 18.24 C ATOM 1258 C ILE 653 1.481 75.547 12.197 1.00 21.88 C ATOM 1259 O ILE 653 2.468 76.273 12.417 1.00 19.90 O ATOM 1260 CB ILE 653 2.728 73.937 10.804 1.00 22.12 C ATOM 1261 CG1 ILE 653 2.805 73.167 9.478 1.00 18.02 C ATOM 1262 CG2 ILE 653 2.957 73.057 12.034 1.00 18.87 C ATOM 1263 CD1 ILE 653 4.217 72.868 9.064 1.00 14.43 C ATOM 1264 N GLY 654 0.445 75.491 13.023 1.00 17.34 N ATOM 1265 CA GLY 654 0.480 76.279 14.245 1.00 18.39 C ATOM 1266 C GLY 654 −0.111 77.682 14.187 1.00 20.92 C ATOM 1267 O GLY 654 −0.373 78.214 13.102 1.00 24.39 O ATOM 1268 N PRO 655 −0.264 78.337 15.363 1.00 20.00 N ATOM 1269 CA PRO 655 −0.806 79.684 15.601 1.00 15.28 C ATOM 1270 C PRO 655 −0.034 80.794 14.911 1.00 16.41 C ATOM 1271 O PRO 655 −0.588 81.825 14.552 1.00 18.28 O ATOM 1272 CB PRO 655 −0.642 79.852 17.116 1.00 12.24 C ATOM 1273 CG PRO 655 −0.672 78.460 17.630 1.00 13.05 C ATOM 1274 CD PRO 655 0.153 77.719 16.635 1.00 14.07 C ATOM 1275 N HIS 656 1.272 80.622 14.794 1.00 16.41 N ATOM 1276 CA HIS 656 2.082 81.667 14.194 1.00 18.52 C ATOM 1277 C HIS 656 2.109 81.703 12.664 1.00 17.35 C ATOM 1278 O HIS 656 2.603 82.672 12.084 1.00 17.65 O ATOM 1279 CB HIS 656 3.492 81.676 14.815 1.00 16.60 C ATOM 1280 CG HIS 656 3.498 82.026 16.283 1.00 18.39 C ATOM 1281 ND1 HIS 656 3.392 81.077 17.277 1.00 18.56 N ATOM 1282 CD2 HIS 656 3.554 83.224 16.915 1.00 16.46 C ATOM 1283 CE1 HIS 656 3.376 81.672 18.451 1.00 13.19 C ATOM 1284 NE2 HIS 656 3.474 82.972 18.258 1.00 16.12 N ATOM 1285 N ALA 657 1.504 80.703 12.027 1.00 17.30 N ATOM 1286 CA ALA 657 1.459 80.616 10.564 1.00 18.06 C ATOM 1287 C ALA 657 0.729 81.810 9.961 1.00 16.82 C ATOM 1288 O ALA 657 −0.300 82.248 10.464 1.00 19.76 O ATOM 1289 CB ALA 657 0.808 79.297 10.121 1.00 13.90 C ATOM 1290 N ASN 658 1.277 82.340 8.877 1.00 19.53 N ATOM 1291 CA ASN 658 0.699 83.502 8.226 1.00 16.37 C ATOM 1292 C ASN 658 −0.569 83.144 7.494 1.00 18.85 C ATOM 1293 O ASN 658 −0.575 82.964 6.284 1.00 20.50 O ATOM 1294 CB ASN 658 1.704 84.142 7.276 1.00 11.24 C ATOM 1295 CG ASN 658 1.282 85.534 6.844 1.00 11.66 C ATOM 1296 OD1 ASN 658 0.098 85.829 6.766 1.00 14.75 O ATOM 1297 ND2 ASN 658 2.251 86.396 6.563 1.00 16.32 N ATOM 1298 N LEU 659 −1.648 83.035 8.251 1.00 20.82 N ATOM 1299 CA LEU 659 −2.943 82.704 7.700 1.00 19.30 C ATOM 1300 C LEU 659 −3.460 83.699 6.664 1.00 20.85 C ATOM 1301 O LEU 659 −4.230 83.317 5.782 1.00 22.69 O ATOM 1302 CB LEU 659 −3.950 82.569 8.824 1.00 17.74 C ATOM 1303 CG LEU 659 −4.579 81.198 8.897 1.00 21.02 C ATOM 1304 CD1 LEU 659 −3.505 80.188 9.248 1.00 24.59 C ATOM 1305 CD2 LEU 659 −5.665 81.212 9.935 1.00 27.06 C ATOM 1306 N LYS 660 −3.078 84.968 6.782 1.00 22.50 N ATOM 1307 CA LYS 660 −3.520 85.982 5.822 1.00 23.11 C ATOM 1308 C LYS 660 −2.945 85.639 4.452 1.00 24.26 C ATOM 1309 O LYS 660 −3.663 85.697 3.448 1.00 27.91 O ATOM 1310 CB LYS 660 −3.068 87.361 6.251 1.00 21.43 C ATOM 1311 N GLN 661 −1.667 85.240 4.420 1.00 21.76 N ATOM 1312 CA GLN 661 −0.991 84.844 3.183 1.00 17.85 C ATOM 1313 C GLN 661 −1.570 83.531 2.688 1.00 18.55 C ATOM 1314 O GLN 661 −1.828 83.372 1.499 1.00 23.06 O ATOM 1315 CB GLN 661 0.525 84.730 3.377 1.00 12.86 C ATOM 1316 CG GLN 661 1.237 86.102 3.366 1.00 10.63 C ATOM 1317 CD GLN 661 2.755 86.013 3.544 1.00 14.73 C ATOM 1318 OE1 GLN 661 3.287 84.965 3.905 1.00 17.21 O ATOM 1319 NE2 GLN 661 3.451 87.118 3.305 1.00 15.89 N ATOM 1320 N ILE 662 −1.850 82.613 3.607 1.00 17.78 N ATOM 1321 CA ILE 662 −2.438 81.336 3.237 1.00 15.00 C ATOM 1322 C ILE 662 −3.775 81.610 2.537 1.00 19.04 C ATOM 1323 O ILE 662 −4.050 81.031 1.487 1.00 21.90 O ATOM 1324 CB ILE 662 −2.580 80.411 4.489 1.00 11.85 C ATOM 1325 CG1 ILE 662 −1.196 80.040 4.995 1.00 11.79 C ATOM 1326 CG2 ILE 662 −3.337 79.126 4.196 1.00 7.88 C ATOM 1327 CD1 ILE 662 −1.191 79.084 6.171 1.00 13.32 C ATOM 1328 N ARG 663 −4.558 82.560 3.047 1.00 20.47 N ATOM 1329 CA ARG 663 −5.850 82.874 2.436 1.00 20.44 C ATOM 1330 C ARG 663 −5.704 83.569 1.078 1.00 19.11 C ATOM 1331 O ARG 663 −6.548 83.416 0.195 1.00 19.37 O ATOM 1332 CB ARG 663 −6.733 83.701 3.390 1.00 23.96 C ATOM 1333 CG ARG 663 −7.123 82.987 4.686 1.00 32.86 C ATOM 1334 CD ARG 663 −7.800 81.612 4.456 1.00 41.17 C ATOM 1335 NE ARG 663 −7.818 80.785 5.672 1.00 43.59 N ATOM 1336 CZ ARG 663 −8.042 79.471 5.708 1.00 44.43 C ATOM 1337 NH1 ARG 663 −8.287 78.791 4.604 1.00 40.35 N ATOM 1338 NH2 ARG 663 −7.942 78.818 6.853 1.00 47.33 N ATOM 1339 N LEU 664 −4.641 84.338 0.911 1.00 20.67 N ATOM 1340 CA LEU 664 −4.400 85.016 −0.351 1.00 23.77 C ATOM 1341 C LEU 664 −4.006 83.990 −1.425 1.00 24.53 C ATOM 1342 O LEU 664 −4.470 84.066 −2.570 1.00 25.26 O ATOM 1343 CB LEU 664 −3.287 86.048 −0.200 1.00 22.42 C ATOM 1344 CG LEU 664 −3.695 87.458 0.188 1.00 23.55 C ATOM 1345 CD1 LEU 664 −2.434 88.240 0.463 1.00 22.20 C ATOM 1346 CD2 LEU 664 −4.517 88.113 −0.910 1.00 21.42 C ATOM 1347 N ILE 665 −3.147 83.040 −1.053 1.00 23.53 N ATOM 1348 CA ILE 665 −2.697 82.009 −1.970 1.00 21.00 C ATOM 1349 C ILE 665 −3.895 81.209 −2.462 1.00 22.09 C ATOM 1350 O ILE 665 −4.064 81.029 −3.659 1.00 24.56 O ATOM 1351 CB ILE 665 −1.680 81.078 −1.316 1.00 17.66 C ATOM 1352 CG1 ILE 665 −0.391 81.841 −1.015 1.00 15.92 C ATOM 1353 CG2 ILE 665 −1.375 79.913 −2.234 1.00 18.28 C ATOM 1354 CD1 ILE 665 0.629 81.031 −0.194 1.00 6.96 C ATOM 1355 N GLU 666 −4.732 80.747 −1.549 1.00 23.35 N ATOM 1356 CA GLU 666 −5.917 79.983 −1.918 1.00 19.08 C ATOM 1357 C GLU 666 −6.813 80.616 −2.967 1.00 19.61 C ATOM 1358 O GLU 666 −7.341 79.920 −3.827 1.00 18.02 O ATOM 1359 CB GLU 666 −6.784 79.754 −0.704 1.00 17.71 C ATOM 1360 CG GLU 666 −6.367 78.618 0.172 1.00 26.16 C ATOM 1361 CD GLU 666 −7.515 78.164 1.049 1.00 28.97 C ATOM 1362 OE1 GLU 666 −7.910 78.950 1.944 1.00 22.27 O ATOM 1363 OE2 GLU 666 −8.030 77.036 0.815 1.00 30.15 O ATOM 1364 N LYS 667 −7.009 81.928 −2.878 1.00 21.77 N ATOM 1365 CA LYS 667 −7.908 82.604 −3.800 1.00 27.64 C ATOM 1366 C LYS 667 −7.336 83.042 −5.140 1.00 28.79 C ATOM 1367 O LYS 667 −8.067 83.548 −5.999 1.00 33.07 O ATOM 1368 CB LYS 667 −8.613 83.773 −3.108 1.00 29.02 C ATOM 1369 CG LYS 667 −7.764 84.976 −2.848 1.00 34.25 C ATOM 1370 CD LYS 667 −8.600 86.097 −2.236 1.00 45.36 C ATOM 1371 CE LYS 667 −7.746 87.344 −1.955 1.00 55.46 C ATOM 1372 NZ LYS 667 −8.130 88.070 −0.688 1.00 58.49 N ATOM 1373 N GLN 668 −6.042 82.833 −5.329 1.00 29.18 N ATOM 1374 CA GLN 668 −5.370 83.189 −6.570 1.00 27.43 C ATOM 1375 C GLN 668 −5.720 82.143 −7.648 1.00 27.44 C ATOM 1376 O GLN 668 −5.795 82.459 −8.834 1.00 29.48 O ATOM 1377 CB GLN 668 −3.856 83.236 −6.324 1.00 24.44 C ATOM 1378 CG GLN 668 −3.199 84.560 −6.643 1.00 31.15 C ATOM 1379 CD GLN 668 −3.934 85.764 −6.071 1.00 33.50 C ATOM 1380 OE1 GLN 668 −4.353 86.659 −6.818 1.00 32.99 O ATOM 1381 NE2 GLN 668 −4.054 85.819 −4.747 1.00 30.05 N ATOM 1382 N ALA 669 −5.930 80.895 −7.231 1.00 23.35 N ATOM 1383 CA ALA 669 −6.275 79.816 −8.153 1.00 21.93 C ATOM 1384 C ALA 669 −6.968 78.683 −7.379 1.00 20.53 C ATOM 1385 O ALA 669 −6.590 78.375 −6.267 1.00 22.44 O ATOM 1386 CB ALA 669 −5.010 79.303 −8.854 1.00 21.51 C ATOM 1387 N PRO 670 −7.983 78.041 −7.977 1.00 22.07 N ATOM 1388 CA PRO 670 −8.697 76.959 −7.300 1.00 19.32 C ATOM 1389 C PRO 670 −7.916 75.769 −6.744 1.00 24.31 C ATOM 1390 O PRO 670 −8.338 75.182 −5.735 1.00 22.85 O ATOM 1391 CB PRO 670 −9.740 76.517 −8.344 1.00 19.25 C ATOM 1392 CG PRO 670 −9.251 77.058 −9.633 1.00 18.97 C ATOM 1393 CD PRO 670 −8.649 78.372 −9.251 1.00 22.64 C ATOM 1394 N GLU 671 −6.765 75.444 −7.332 1.00 21.90 N ATOM 1395 CA GLU 671 −6.037 74.282 −6.872 1.00 21.53 C ATOM 1396 C GLU 671 −5.121 74.573 −5.724 1.00 18.85 C ATOM 1397 O GLU 671 −4.526 73.648 −5.152 1.00 17.02 O ATOM 1398 CB GLU 671 −5.237 73.622 −7.996 1.00 28.16 C ATOM 1399 CG GLU 671 −6.053 73.156 −9.167 1.00 30.85 C ATOM 1400 CD GLU 671 −6.052 74.163 −10.295 1.00 40.33 C ATOM 1401 OE1 GLU 671 −5.922 75.389 −10.033 1.00 37.89 O ATOM 1402 OE2 GLU 671 −6.181 73.718 −11.456 1.00 49.39 O ATOM 1403 N ASN 672 −4.915 75.849 −5.440 1.00 16.70 N ATOM 1404 CA ASN 672 −4.035 76.229 −4.324 1.00 20.34 C ATOM 1405 C ASN 672 −4.755 75.885 −3.007 1.00 18.55 C ATOM 1406 O ASN 672 −5.136 76.780 −2.248 1.00 18.65 O ATOM 1407 CB ASN 672 −3.751 77.739 −4.359 1.00 19.53 C ATOM 1408 CG ASN 672 −2.994 78.186 −5.601 1.00 15.33 C ATOM 1409 OD1 ASN 672 −2.466 77.384 −6.369 1.00 12.87 O ATOM 1410 ND2 ASN 672 −2.914 79.494 −5.778 1.00 17.27 N ATOM 1411 N LYS 673 −4.986 74.601 −2.761 1.00 17.76 N ATOM 1412 CA LYS 673 −5.708 74.178 −1.568 1.00 21.07 C ATOM 1413 C LYS 673 −4.820 74.299 −0.335 1.00 20.99 C ATOM 1414 O LYS 673 −3.655 73.896 −0.360 1.00 26.08 O ATOM 1415 CB LYS 673 −6.222 72.732 −1.710 1.00 19.00 C ATOM 1416 CG LYS 673 −7.480 72.469 −0.858 1.00 30.07 C ATOM 1417 CD LYS 673 −7.457 71.176 −0.047 1.00 30.38 C ATOM 1418 CE LYS 673 −7.751 69.983 −0.913 1.00 36.80 C ATOM 1419 NZ LYS 673 −7.645 68.682 −0.181 1.00 45.16 N ATOM 1420 N ALA 674 −5.357 74.891 0.727 1.00 18.75 N ATOM 1421 CA ALA 674 −4.613 75.043 1.959 1.00 15.72 C ATOM 1422 C ALA 674 −4.928 73.914 2.916 1.00 18.41 C ATOM 1423 O ALA 674 −6.047 73.370 2.935 1.00 12.37 O ATOM 1424 CB ALA 674 −4.938 76.362 2.607 1.00 14.82 C ATOM 1425 N PHE 675 −3.916 73.577 3.716 1.00 20.72 N ATOM 1426 CA PHE 675 −4.021 72.560 4.751 1.00 17.35 C ATOM 1427 C PHE 675 −3.565 73.235 6.032 1.00 13.94 C ATOM 1428 O PHE 675 −2.414 73.594 6.162 1.00 16.79 O ATOM 1429 CB PHE 675 −3.148 71.357 4.421 1.00 17.30 C ATOM 1430 CG PHE 675 −3.667 70.547 3.276 1.00 16.73 C ATOM 1431 CD1 PHE 675 −3.357 70.890 1.961 1.00 18.39 C ATOM 1432 CD2 PHE 675 −4.502 69.469 3.501 1.00 11.98 C ATOM 1433 CE1 PHE 675 −3.880 70.177 0.880 1.00 11.40 C ATOM 1434 CE2 PHE 675 −5.031 68.746 2.416 1.00 15.53 C ATOM 1435 CZ PHE 675 −4.717 69.111 1.108 1.00 13.44 C ATOM 1436 N VAL 676 −4.509 73.561 6.899 1.00 17.73 N ATOM 1437 CA VAL 676 −4.176 74.211 8.163 1.00 19.36 C ATOM 1438 C VAL 676 −4.128 73.145 9.260 1.00 20.91 C ATOM 1439 O VAL 676 −5.035 72.324 9.395 1.00 19.66 O ATOM 1440 CB VAL 676 −5.175 75.326 8.489 1.00 17.55 C ATOM 1441 CG1 VAL 676 −4.779 76.031 9.769 1.00 18.65 C ATOM 1442 CG2 VAL 676 −5.219 76.328 7.325 1.00 19.14 C ATOM 1443 N LEU 677 −2.994 73.100 9.952 1.00 23.37 N ATOM 1444 CA LEU 677 −2.740 72.122 11.003 1.00 21.69 C ATOM 1445 C LEU 677 −2.362 72.830 12.284 1.00 17.44 C ATOM 1446 O LEU 677 −1.870 73.965 12.263 1.00 11.94 O ATOM 1447 CB LEU 677 −1.597 71.184 10.603 1.00 25.46 C ATOM 1448 CG LEU 677 −1.766 70.099 9.540 1.00 25.42 C ATOM 1449 CD1 LEU 677 −2.269 70.624 8.236 1.00 23.58 C ATOM 1450 CD2 LEU 677 −0.422 69.495 9.346 1.00 30.67 C ATOM 1451 N SER 678 −2.591 72.153 13.403 1.00 15.75 N ATOM 1452 CA SER 678 −2.279 72.728 14.704 1.00 18.74 C ATOM 1453 C SER 678 −0.805 72.690 15.096 1.00 15.98 C ATOM 1454 O SER 678 −0.305 73.594 15.775 1.00 16.52 O ATOM 1455 CB SER 678 −3.143 72.085 15.783 1.00 17.24 C ATOM 1456 OG SER 678 −4.495 72.476 15.621 1.00 23.02 O ATOM 1457 N SER 679 −0.095 71.679 14.621 1.00 15.13 N ATOM 1458 CA SER 679 1.305 71.550 14.954 1.00 16.64 C ATOM 1459 C SER 679 1.891 70.526 13.993 1.00 16.28 C ATOM 1460 O SER 679 1.152 69.830 13.284 1.00 16.75 O ATOM 1461 CB SER 679 1.424 71.025 16.389 1.00 20.41 C ATOM 1462 OG SER 679 0.924 69.683 16.472 1.00 15.18 O ATOM 1463 N VAL 680 3.212 70.389 14.023 1.00 14.70 N ATOM 1464 CA VAL 680 3.881 69.426 13.165 1.00 18.08 C ATOM 1465 C VAL 680 3.441 67.999 13.506 1.00 21.23 C ATOM 1466 O VAL 680 3.598 67.092 12.692 1.00 22.59 O ATOM 1467 CB VAL 680 5.421 69.505 13.291 1.00 15.16 C ATOM 1468 CG1 VAL 680 5.953 70.852 12.792 1.00 14.14 C ATOM 1469 CG2 VAL 680 5.844 69.260 14.728 1.00 15.78 C ATOM 1470 N ASP 681 2.845 67.803 14.680 1.00 23.61 N ATOM 1471 CA ASP 681 2.431 66.465 15.090 1.00 20.29 C ATOM 1472 C ASP 681 1.267 65.967 14.292 1.00 17.46 C ATOM 1473 O ASP 681 1.021 64.758 14.254 1.00 19.43 O ATOM 1474 CB ASP 681 2.118 66.404 16.586 1.00 22.71 C ATOM 1475 CG ASP 681 3.360 66.605 17.455 1.00 27.60 C ATOM 1476 OD1 ASP 681 4.485 66.251 17.021 1.00 31.79 O ATOM 1477 OD2 ASP 681 3.205 67.117 18.585 1.00 30.83 O ATOM 1478 N GLU 682 0.559 66.891 13.648 1.00 19.92 N ATOM 1479 CA GLU 682 −0.593 66.532 12.823 1.00 19.43 C ATOM 1480 C GLU 682 −0.246 66.200 11.373 1.00 16.94 C ATOM 1481 O GLU 682 −1.031 65.593 10.678 1.00 20.34 O ATOM 1482 CB GLU 682 −1.664 67.616 12.904 1.00 19.71 C ATOM 1483 CG GLU 682 −2.031 67.903 14.337 1.00 27.72 C ATOM 1484 CD GLU 682 −3.385 68.541 14.516 1.00 32.65 C ATOM 1485 OE1 GLU 682 −3.806 69.338 13.643 1.00 32.50 O ATOM 1486 OE2 GLU 682 −4.013 68.250 15.567 1.00 36.39 O ATOM 1487 N LEU 683 0.968 66.533 10.953 1.00 19.46 N ATOM 1488 CA LEU 683 1.430 66.270 9.591 1.00 18.53 C ATOM 1489 C LEU 683 1.230 64.820 9.159 1.00 19.54 C ATOM 1490 O LEU 683 0.759 64.567 8.050 1.00 21.33 O ATOM 1491 CB LEU 683 2.913 66.629 9.458 1.00 9.01 C ATOM 1492 CG LEU 683 3.212 68.119 9.485 1.00 11.07 C ATOM 1493 CD1 LEU 683 4.725 68.380 9.521 1.00 6.20 C ATOM 1494 CD2 LEU 683 2.581 68.729 8.268 1.00 9.94 C ATOM 1495 N GLU 684 1.577 63.879 10.027 1.00 17.82 N ATOM 1496 CA GLU 684 1.449 62.470 9.701 1.00 21.58 C ATOM 1497 C GLU 684 0.026 62.043 9.404 1.00 20.80 C ATOM 1498 O GLU 684 −0.204 61.216 8.521 1.00 21.28 O ATOM 1499 CB GLU 684 2.022 61.614 10.826 1.00 27.44 C ATOM 1500 CG GLU 684 1.799 60.112 10.601 1.00 42.61 C ATOM 1501 CD GLU 684 2.605 59.219 11.539 1.00 48.25 C ATOM 1502 OE1 GLU 684 2.910 59.655 12.683 1.00 46.17 O ATOM 1503 OE2 GLU 684 2.915 58.071 11.116 1.00 50.28 O ATOM 1504 N GLN 685 −0.914 62.632 10.133 1.00 22.35 N ATOM 1505 CA GLN 685 −2.349 62.353 10.005 1.00 25.85 C ATOM 1506 C GLN 685 −2.958 62.892 8.709 1.00 22.14 C ATOM 1507 O GLN 685 −4.089 62.579 8.381 1.00 25.03 O ATOM 1508 CB GLN 685 −3.117 63.008 11.162 1.00 25.71 C ATOM 1509 CG GLN 685 −2.414 62.982 12.509 1.00 34.50 C ATOM 1510 CD GLN 685 −3.192 63.753 13.571 1.00 41.31 C ATOM 1511 OE1 GLN 685 −4.338 64.167 13.356 1.00 40.86 O ATOM 1512 NE2 GLN 685 −2.565 63.957 14.726 1.00 45.33 N ATOM 1513 N GLN 686 −2.245 63.789 8.044 1.00 20.96 N ATOM 1514 CA GLN 686 −2.715 64.404 6.813 1.00 23.16 C ATOM 1515 C GLN 686 −1.882 64.075 5.583 1.00 19.32 C ATOM 1516 O GLN 686 −2.328 64.299 4.456 1.00 22.21 O ATOM 1517 CB GLN 686 −2.709 65.912 6.982 1.00 26.35 C ATOM 1518 CG GLN 686 −3.709 66.416 7.948 1.00 35.22 C ATOM 1519 CD GLN 686 −4.816 67.142 7.241 1.00 42.30 C ATOM 1520 OE1 GLN 686 −4.941 68.365 7.350 1.00 45.54 O ATOM 1521 NE2 GLN 686 −5.616 66.400 6.478 1.00 44.88 N ATOM 1522 N ARG 687 −0.683 63.557 5.803 1.00 14.84 N ATOM 1523 CA ARG 687 0.238 63.225 4.731 1.00 17.34 C ATOM 1524 C ARG 687 −0.364 62.511 3.524 1.00 17.95 C ATOM 1525 O ARG 687 −0.248 62.982 2.402 1.00 18.16 O ATOM 1526 CB ARG 687 1.405 62.429 5.303 1.00 12.76 C ATOM 1527 CG ARG 687 2.512 62.076 4.320 1.00 12.31 C ATOM 1528 CD ARG 687 2.812 60.589 4.398 1.00 15.47 C ATOM 1529 NE ARG 687 2.836 60.134 5.786 1.00 27.33 N ATOM 1530 CZ ARG 687 2.343 58.985 6.234 1.00 25.36 C ATOM 1531 NH1 ARG 687 1.783 58.118 5.416 1.00 25.62 N ATOM 1532 NH2 ARG 687 2.342 58.744 7.535 1.00 34.14 N ATOM 1533 N ASP 688 −1.044 61.397 3.749 1.00 22.35 N ATOM 1534 CA ASP 688 −1.603 60.649 2.632 1.00 20.59 C ATOM 1535 C ASP 688 −2.651 61.418 1.872 1.00 23.19 C ATOM 1536 O ASP 688 −2.732 61.326 0.653 1.00 23.82 O ATOM 1537 CB ASP 688 −2.128 59.301 3.096 1.00 21.54 C ATOM 1538 CG ASP 688 −1.008 58.365 3.518 1.00 24.25 C ATOM 1539 OD1 ASP 688 0.172 58.697 3.266 1.00 31.52 O ATOM 1540 OD2 ASP 688 −1.287 57.289 4.083 1.00 26.17 O ATOM 1541 N GLU 689 −3.395 62.253 2.575 1.00 23.99 N ATOM 1542 CA GLU 689 −4.419 63.045 1.927 1.00 26.49 C ATOM 1543 C GLU 689 −3.796 64.142 1.054 1.00 25.62 C ATOM 1544 O GLU 689 −4.376 64.536 0.034 1.00 26.10 O ATOM 1545 CB GLU 689 −5.334 63.663 2.981 1.00 34.19 C ATOM 1546 CG GLU 689 −6.456 64.543 2.429 1.00 36.12 C ATOM 1547 CD GLU 689 −7.358 65.061 3.527 1.00 42.19 C ATOM 1548 OE1 GLU 689 −7.532 64.327 4.532 1.00 50.63 O ATOM 1549 OE2 GLU 689 −7.895 66.189 3.385 1.00 42.61 O ATOM 1550 N ILE 690 −2.645 64.665 1.478 1.00 21.08 N ATOM 1551 CA ILE 690 −1.951 65.705 0.726 1.00 19.71 C ATOM 1552 C ILE 690 −1.312 65.124 −0.514 1.00 20.60 C ATOM 1553 O ILE 690 −1.368 65.722 −1.567 1.00 23.29 O ATOM 1554 CB ILE 690 −0.881 66.388 1.572 1.00 20.01 C ATOM 1555 CG1 ILE 690 −1.550 67.130 2.737 1.00 18.80 C ATOM 1556 CG2 ILE 690 −0.054 67.322 0.713 1.00 19.33 C ATOM 1557 CD1 ILE 690 −0.610 67.889 3.623 1.00 13.89 C ATOM 1558 N VAL 691 −0.694 63.958 −0.394 1.00 22.47 N ATOM 1559 CA VAL 691 −0.083 63.301 −1.543 1.00 21.73 C ATOM 1560 C VAL 691 −1.144 62.902 −2.579 1.00 20.40 C ATOM 1561 O VAL 691 −0.923 63.076 −3.767 1.00 22.98 O ATOM 1562 CB VAL 691 0.750 62.083 −1.110 1.00 22.75 C ATOM 1563 CG1 VAL 691 1.147 61.263 −2.296 1.00 29.43 C ATOM 1564 CG2 VAL 691 1.988 62.555 −0.431 1.00 23.51 C ATOM 1565 N SER 692 −2.296 62.407 −2.139 1.00 17.46 N ATOM 1566 CA SER 692 −3.371 62.031 −3.054 1.00 15.69 C ATOM 1567 C SER 692 −3.894 63.216 −3.856 1.00 16.29 C ATOM 1568 O SER 692 −4.106 63.117 −5.060 1.00 19.86 O ATOM 1569 CB SER 692 −4.532 61.435 −2.283 1.00 15.65 C ATOM 1570 OG SER 692 −4.134 60.230 −1.674 1.00 28.15 O ATOM 1571 N TYR 693 −4.129 64.319 −3.168 1.00 16.74 N ATOM 1572 CA TYR 693 −4.632 65.536 −3.768 1.00 15.99 C ATOM 1573 C TYR 693 −3.654 65.960 −4.833 1.00 18.79 C ATOM 1574 O TYR 693 −3.983 66.084 −6.006 1.00 21.36 O ATOM 1575 CB TYR 693 −4.725 66.619 −2.702 1.00 11.55 C ATOM 1576 CG TYR 693 −5.078 67.969 −3.259 1.00 19.55 C ATOM 1577 CD1 TYR 693 −6.374 68.238 −3.748 1.00 20.06 C ATOM 1578 CD2 TYR 693 −4.102 68.960 −3.394 1.00 13.67 C ATOM 1579 CE1 TYR 693 −6.665 69.458 −4.365 1.00 14.61 C ATOM 1580 CE2 TYR 693 −4.394 70.165 −4.002 1.00 14.06 C ATOM 1581 CZ TYR 693 −5.664 70.402 −4.489 1.00 14.63 C ATOM 1582 OH TYR 693 −5.911 71.592 −5.123 1.00 17.27 O ATOM 1583 N LEU 694 −2.416 66.070 −4.401 1.00 21.64 N ATOM 1584 CA LEU 694 −1.287 66.480 −5.216 1.00 22.01 C ATOM 1585 C LEU 694 −1.001 65.549 −6.414 1.00 24.72 C ATOM 1586 O LEU 694 −0.819 66.007 −7.560 1.00 21.88 O ATOM 1587 CB LEU 694 −0.131 66.631 −4.229 1.00 19.20 C ATOM 1588 CG LEU 694 1.365 66.561 −4.388 1.00 23.50 C ATOM 1589 CD1 LEU 694 1.939 67.326 −3.227 1.00 20.23 C ATOM 1590 CD2 LEU 694 1.840 65.126 −4.387 1.00 21.48 C ATOM 1591 N CYS 695 −1.033 64.243 −6.178 1.00 24.01 N ATOM 1592 CA CYS 695 −0.805 63.264 −7.230 1.00 23.59 C ATOM 1593 C CYS 695 −1.913 63.363 −8.266 1.00 23.92 C ATOM 1594 O CYS 695 −1.662 63.443 −9.476 1.00 20.77 O ATOM 1595 CB CYS 695 −0.785 61.851 −6.639 1.00 21.84 C ATOM 1596 SG CYS 695 −0.122 60.600 −7.770 1.00 25.31 S ATOM 1597 N ASP 696 −3.141 63.401 −7.775 1.00 25.04 N ATOM 1598 CA ASP 696 −4.312 63.463 −8.634 1.00 30.16 C ATOM 1599 C ASP 696 −4.295 64.617 −9.630 1.00 29.12 C ATOM 1600 O ASP 696 −4.933 64.557 −10.678 1.00 33.49 O ATOM 1601 CB ASP 696 −5.569 63.536 −7.775 1.00 33.29 C ATOM 1602 CG ASP 696 −6.827 63.645 −8.598 1.00 40.85 C ATOM 1603 OD1 ASP 696 −7.215 62.641 −9.244 1.00 44.44 O ATOM 1604 OD2 ASP 696 −7.416 64.749 −8.613 1.00 45.63 O ATOM 1605 N LEU 697 −3.544 65.655 −9.296 1.00 27.97 N ATOM 1606 CA LEU 697 −3.421 66.863 −10.108 1.00 23.65 C ATOM 1607 C LEU 697 −2.498 66.673 −11.297 1.00 21.19 C ATOM 1608 O LEU 697 −2.676 67.307 −12.337 1.00 23.84 O ATOM 1609 CB LEU 697 −2.863 67.990 −9.227 1.00 19.78 C ATOM 1610 CG LEU 697 −3.679 69.216 −8.812 1.00 18.30 C ATOM 1611 CD1 LEU 697 −5.186 68.949 −8.767 1.00 12.42 C ATOM 1612 CD2 LEU 697 −3.128 69.715 −7.478 1.00 11.15 C ATOM 1613 N ALA 698 −1.467 65.864 −11.106 1.00 20.21 N ATOM 1614 CA ALA 698 −0.472 65.597 −12.128 1.00 18.13 C ATOM 1615 C ALA 698 −0.972 64.732 −13.286 1.00 18.60 C ATOM 1616 O ALA 698 −1.904 63.935 −13.128 1.00 20.41 O ATOM 1617 CB ALA 698 0.746 64.965 −11.481 1.00 16.85 C ATOM 1618 N PRO 699 −0.362 64.887 −14.479 1.00 15.84 N ATOM 1619 CA PRO 699 −0.802 64.081 −15.622 1.00 14.86 C ATOM 1620 C PRO 699 −0.313 62.649 −15.472 1.00 17.66 C ATOM 1621 O PRO 699 0.687 62.397 −14.808 1.00 18.33 O ATOM 1622 CB PRO 699 −0.131 64.763 −16.817 1.00 10.16 C ATOM 1623 CG PRO 699 0.224 66.151 −16.293 1.00 11.52 C ATOM 1624 CD PRO 699 0.636 65.888 −14.889 1.00 12.64 C ATOM 1625 N GLU 700 −1.029 61.702 −16.058 1.00 18.23 N ATOM 1626 CA GLU 700 −0.585 60.329 −15.996 1.00 21.35 C ATOM 1627 C GLU 700 0.318 60.141 −17.171 1.00 24.65 C ATOM 1628 O GLU 700 0.213 60.873 −18.137 1.00 29.66 O ATOM 1629 CB GLU 700 −1.730 59.377 −16.176 1.00 19.18 C ATOM 1630 CG GLU 700 −2.640 59.327 −15.036 1.00 27.76 C ATOM 1631 CD GLU 700 −3.623 58.221 −15.210 1.00 30.98 C ATOM 1632 OE1 GLU 700 −3.184 57.061 −15.339 1.00 35.58 O ATOM 1633 OE2 GLU 700 −4.833 58.511 −15.269 1.00 39.02 O ATOM 1634 N ALA 701 1.223 59.173 −17.083 1.00 33.25 N ATOM 1635 CA ALA 701 2.109 58.864 −18.203 1.00 34.46 C ATOM 1636 C ALA 701 1.251 58.146 −19.254 1.00 36.32 C ATOM 1637 O ALA 701 0.277 57.456 −18.916 1.00 30.64 O ATOM 1638 CB ALA 701 3.251 57.962 −17.739 1.00 32.72 C ATOM 1639 N PRO 702 1.537 58.370 −20.545 1.00 42.71 N ATOM 1640 CA PRO 702 0.790 57.732 −21.634 1.00 47.12 C ATOM 1641 C PRO 702 1.089 56.237 −21.591 1.00 49.56 C ATOM 1642 O PRO 702 2.244 55.851 −21.342 1.00 48.33 O ATOM 1643 CB PRO 702 1.402 58.353 −22.886 1.00 45.52 C ATOM 1644 CG PRO 702 1.918 59.687 −22.387 1.00 48.78 C ATOM 1645 CD PRO 702 2.530 59.322 −21.073 1.00 44.35 C ATOM 1646 N PRO 703 0.080 55.383 −21.849 1.00 51.95 N ATOM 1647 CA PRO 703 0.189 53.922 −21.841 1.00 54.88 C ATOM 1648 C PRO 703 1.480 53.458 −22.521 1.00 59.06 C ATOM 1649 O PRO 703 1.819 53.929 −23.616 1.00 56.81 O ATOM 1650 CB PRO 703 −1.057 53.477 −22.606 1.00 53.66 C ATOM 1651 CG PRO 703 −2.041 54.516 −22.235 1.00 53.29 C ATOM 1652 CD PRO 703 −1.240 55.790 −22.379 1.00 53.11 C ATOM 1653 N PRO 704 2.271 52.622 −21.816 1.00 64.17 N ATOM 1654 CA PRO 704 3.533 52.117 −22.370 1.00 66.20 C ATOM 1655 C PRO 704 3.250 51.526 −23.741 1.00 67.33 C ATOM 1656 O PRO 704 2.480 50.565 −23.877 1.00 66.63 O ATOM 1657 CB PRO 704 3.946 51.050 −21.356 1.00 66.86 C ATOM 1658 CG PRO 704 3.457 51.639 −20.067 1.00 65.74 C ATOM 1659 CD PRO 704 2.065 52.108 −20.449 1.00 64.51 C ATOM 1660 N THR 705 3.885 52.118 −24.741 1.00 67.88 N ATOM 1661 CA THR 705 3.712 51.735 −26.130 1.00 68.43 C ATOM 1662 C THR 705 5.009 51.114 −26.688 1.00 68.19 C ATOM 1663 O THR 705 4.932 50.046 −27.340 1.00 66.67 O ATOM 1664 CB THR 705 3.231 52.998 −26.933 1.00 69.60 C ATOM 1665 OG1 THR 705 2.672 52.619 −28.198 1.00 70.05 O ATOM 1666 CG2 THR 705 4.357 54.013 −27.124 1.00 68.20 C ATOM 1667 OXT THR 705 6.102 51.650 −26.404 1.00 67.96 O

To demonstrate the feasibility of identifying potential small molecule inhibitors in silico, computational modeling software was utilized in conjunction with high-resolution crystal structure results to screen databases for existing compounds that would bind to the A1 domain where it interfaces with botrocetin (exogenous ligand binding site). Several small molecules predicted to bind with sub-micromolar IC₅₀s (concentration of drug required to inhibit the activity by 50%) and that could also severely disrupt binding of this snake venom protein were identified. Thus, potential candidate small molecules can be identified that may interfere with the interaction between GPIb alpha and the A1 domain of VWF.

Screening small molecule library for inhibitors. Although the use of computational modeling is a state-of-the-art method for identifying lead compounds, it is not without its limitations. Thus, we will also screen an actual library of 20,000 small molecules manufactured by the Chembridge Corporation (San Diego, Calif.). The library consists of handcrafted drug-like organic molecules with molecular weights in a range of 25-550, which are soluble in DMSO at concentrations ranging from 10-20 mM. The structure and purity (>95%) of these compounds have been validated by NMR. The library is formatted in a 96 well plate for high throughput screening using instrumentation made available through the OCCC (under supervision of the Landry laboratory) and includes a robot plate reader (FLexStation II 384, Molecular Devices, Sunnyvale, Calif.), an 8-tip robotic pipettor (Multiprobe II Plus, Perkin Elmer, Shelton, Conn.), a 96-tip robotic pipettor (Mintrak, Perkin Elmer), and an automated 96 well plate washer (Perkin Elmer).

An ELISA based system will be used to screen for compounds that may inhibit the interaction between GPIb alpha and the VWF-A1 domain. Enzyme-Linked Immunosorbent Assay (ELISA) methods are immunoassay techniques used for detection or quantification of a substance. An example of this assay is demonstrated in FIG. 45A, where an antibody conjugated with horseradish peroxidase (HRP) was used to identify the presence of VWF. Depending on the substrate added, HRP enzyme activity can be detected by either a change in color (chromogenic product) or fluorescence (most sensitive indicator). Schematic representation of the proposed assay system to be used for screening is shown in FIG. 45A.

Assay system: Recombinant GPIb alpha and VWF-A1 proteins will be generated and purified as described in the attached articles, with the latter containing a 6×His tag. Purified GPIb alpha will be absorbed overnight (4° C.) to PRO-BIND polystyrene 96-well assay plates (Falcon) at 10 μg/ml per well. Plates will be washed and non-specific binding sites blocked by the addition of TENTC buffer (50 mM Tris, 1 mM EDTA, 0.15 M NaCl, 0.2% casein, 0.05% Tween 20, pH 8.0) for 1 hour at room temperature. Subsequently, plates will be washed with and resuspended in TBS buffer (50 mM Tris, 150 mM NaCl, pH 8.0) and 1 test compound per well added at a final concentration of 10 μM (final DMSO concentration 0.5%). After 30 min, recombinant His tagged VWF-A1 protein will be added at a 1:1 Molar ratio to that of GPIb alpha and left to incubate for 1 hour before washing with TBS buffer. VWF-A1 bound to surface-immobilized GPIb alpha will be determined by the addition of HRP-conjugated anti-His tag antibody and the A1-antibody conjugate detected by the addition of LumiGlow reagent (KPL, Gaithersburg, Md.). The resulting fluorescence will be quantified by of the number of luminescence emissions per second using a FLexStation II 384 plate reader. A sample will be considered positive when the luminescence (in counts per second) is more than 2 standard deviations above the mean value for negative-controls.

Negative controls: Addition of mAb 6D1 to certain wells to prevent VWF-A1 binding to GPIb alpha or no addition of VWF-A1 protein (FIG. 45B). In either case, no significant fluorescence should be detected. Once compounds of interest have been identified, solubility of these molecules will be confirmed to rule out precipitation as the etiology for blocking interactions between GPIb alpha and VWF-A1. In addition, a dose effect curve will also be generated (1 nM to 100 μM) to obtain preliminary information regarding the IC50 of the inhibitor. Lead molecules will then be tested for their ability to limit human platelet interactions with plasma VWF in aggregometry and flow chamber assays as described in preliminary results. Ultimately, the most promising compound will be tested in our humanized mouse model of thrombosis.

Example 7 Effect of Plavix or ReoPro on Human Platelet-Induced Hemostasis in Homozygous VWF^(1326R>H) Mice

To demonstrate the feasibility of our VWF^(1326R>H) mice to identify anti-thrombotic drugs capable of perturbing human platelet function in vivo, we tested the ability of 2 FDA approved drugs, Plavix and ReoPro, to prevent human platelet-induced hemostasis. Plavix is the second most commonly used anti-thrombotic drug that targets one of the ADP receptors (P2Y12) on platelets, causing irreversible inhibition (Hankey et al. Med. J. Aust. 2003; 178:568). ADP is a potent mediator of platelet activation and aggregate formation, and thus considerable effort and funds have been devoted to inhibiting this activation pathway in platelets. Clopidogrel was approved by the FDA in 1997 for clinical use and was found to be of benefit in the secondary prevention of major vascular events in patients with a history of cerebrovascular and coronary artery diseases and major cardiac events post coronary artery stent placement (Gachet et al. Semin. Thromb. Hemost. 2005; 31:162). Disadvantages of this drug are: 1) It must be metabolized in the liver to generate an active metabolite, thus limiting its effectiveness in acute settings, and 2) irreversible inhibition that results in a marked prolongation of bleeding time. Clopidogrel has been shown to reduce thrombus size and delay its formation in mice with a maximal effective dose of 50 mg/kg given the day before and 2 hours prior to experimentation. Homozygous VWF^(1326R>H) mice that received this dosing schema, were unable to produce a hemostatic clot when administered human platelets in contrast to homozygous VWF^(1326R>H) mice that received saline in lieu of drug.

As Plavix can also block the function of the ADP receptor on murine platelets (see FIG. 46A), we also tested the ability of ReoPro to prevent the formation of a hemostatic plug in homozygous VWF^(1326R>H) mice. ReoPro is a Fab fragment of a human-murine chimeric monoclonal antibody that blocks fibrinogen binding to the platelet integrin receptor αIIbβ3, thus limiting thrombus growth (Bennett, J. S. Novel platelet inhibitors. Annu. Rev. Med. 52, 161-184 (2001)). It is currently approved for short-term treatment of patients with acute coronary syndrome that require interventional catheterization. It is administered by intravenously bolus (0.25 mg/kg), followed by an infusion of 0.125 μg/kg/min. This results in >80% αIIbβ3 occupancy, and disrupts platelet function for 24-36 h. It does not bind or disrupt the function of murine αIIbβ3. Administration of ReoPro to homozygous VWF^(1326R>H) mice 5 minutes after the infusion of human platelets, prevented the formation of a hemostatic plug (mean bleeding time 579 sec) (FIG. 46B). By contrast, animals that received a non-function blocking antibody to human αIIbβ3 were able to form a hemostatic plug (mean bleeding 175 sec).

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Example 8 Determining the Efficacy of Anti-Platelet Drugs Administered to Patients by Studying the Ability of Platelets Harvested from Patients on Therapies in the VWF^(1326R>H) Mouse

Intravital microscopic study was carried out to evaluate the ability of the VWF^(1326R>H) mouse to determine the efficacy of anti-platelet therapies given to patients at risk or with active cardiovascular disease. The typical prophylactic dose of aspirin (ASA) of 81 mg did not prevent laser-injury induced human platelet thrombus formation in the genetically modified animal while increasing the daily dose to 162 mg was preventative (FIG. 47). Similarly, platelets administered from a patient on 81 mg of ASA and 75 mg Plavix also prevented thrombus formation. 

1. An isolated mutant human von Willebrand Factor A1 protein comprising one or more selected from the group consisting of: 1263 S>P, 1269D>N, 1274R>K, 1287R>M, 1302D>G, 1308R>H, 1313W>R, 1314V>I, 1326H>R, 1329I>L, 1330G>E, 1333D>A, 1344A>T, 1347V>I, 1350A>T, 1370S>G, 1379R>H, 1381A>T, 1385M>T 1391Q>P, 1394S>A, 1397F>L, 1421N>S, 1439V>L, 1442S>G, 1449Q>R, 1466P>A, 1469L>Q, 1472H>Q, 1473M>V, 1475Q>H, and 1479G>S, wherein each amino acid position corresponds to a position in SEQ ID NO:
 6. 2. An isolated mutant human von Willebrand Factor A1 protein having SEQ ID NO: 6, wherein the protein comprises one or more mutation(s) selected from the group consisting of: 1263S>P, 1269D>N, 1274R>K, 1287R>M, 1302D>G, 1308R>H, 1313W>R, 1314V>I, 1326H>R, 1329I>L, 1330G>E, 1333D>A, 1344A>T, 1347V>I, 1350A>T, 1370S>G, 1379R>H, 1381A>T, 1385M>T, 1391Q>P, 1394S>A, 1397F>L, 1421N>S, 1439V>L, 1442S>G, 1449Q>R, 1466P>A, 1469L>Q, 1472H>Q, 1473M>V, 1475Q>H, and a 1479G>S.
 3. An isolated mutant human von Willebrand Factor A1 protein comprising a 1326H>R mutation in an amino acid sequence of SEQ ID NO:
 1. 4. A transgenic non-human animal expressing von Willebrand Factor A1 protein containing mutation(s) at one of more amino acid position selected from the group consisting of: 1263, 1269, 1274, 1287, 1302, 1308, 1313, 1314, 1326, 1329, 1330, 1333, 1344, 1347, 1350, 1370, 1379, 1381, 1385, 1391, 1394, 1397, 1421, 1439, 1442, 1449, 1466, 1469, 1472, 1473, 1475, and 1479, wherein the position corresponds to an amino acid position of human von Willebrand Factor A1 protein shown in SEQ ID NO:
 6. 5. The transgenic non-human animal of claim 4, wherein the animal is a murine, a porcine, a canine, a feline, a rabbit, or a primate.
 6. The transgenic non-human animal of claim 4, wherein the protein comprises a single mutation.
 7. The transgenic non-human animal of claim 4, wherein the protein comprises two or more mutations.
 8. The transgenic non-human animal of claim 4, wherein the protein comprises at least one mutation selected from the group consisting of: 1263>S, 1269>D, 1274>R, 1287>R, 1302>D, 1308>R, 1313R>W, 1314>V, 1326>H, 1329>I, 1330>G, 1333>D, 1344>A, 1347>V, 1350>A, 1370>S, 1379>R, 1381>A, 1385>M 1391>Q, 1394>S, 1397>F, 1421>N, 1439>V, 1442>S, 1449>Q, 1466>P, 1469>L, 1472>H, 1473>M, 1475>Q, 1479>G, and any combination thereof.
 9. The transgenic non-human animal of claim 4, wherein the protein comprises a 1326R>H mutation, a 1314I>V mutation, or a combination thereof.
 10. The transgenic non-human animal of claim 8 or 9, wherein the animal is a mouse.
 11. The transgenic non-human animal of claim 8 or 9, wherein the protein comprises SEQ ID NO:
 5. 12. The transgenic non-human animal of claim 4, wherein the VWF protein is at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the A1 domain of human VWF protein as shown in SEQ ID NO:
 1. 13. The transgenic non-human animal of claim 12, wherein the von Willebrand Factor A1 protein of the transgenic animal contains the human A1 domain shown in SEQ ID NO:
 1. 14. The transgenic non-human animal of claim 4, wherein the von Willebrand Factor A1 protein is partially or completely replaced with a human von Willebrand Factor A1 protein comprising SEQ ID NO:
 1. 15. The transgenic non-human animal of claim 4, wherein the animal is a model for pre-clinical testing of compounds that expresses a mutant von Willebrand Factor (VWF) A1 protein containing one or more mutations, wherein the binding specificity of the mutant VWF-A1 protein changes from being specific for the animal platelets to being specific for human platelets.
 16. The transgenic non-human animal of claim 15, wherein the mutant VWF-A1 protein in the animal binds to human platelets.
 17. A method for identifying a compound that modulates binding of VWF-A1 protein to GPIb-alpha protein, the method comprising: a) providing an electronic library of test compounds; b) providing atomic coordinates listed in Table 8 for at least 10 amino acid residues for the A1 domain of the VWF protein, wherein the coordinates have a root mean square deviation therefrom, with respect to at least 50% of Cα atoms, of not greater than about 2.5 Å, in a computer readable format; c) converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the VWF-A1 domain; d) performing a data processing method, wherein electronic test compounds from the library are superimposed upon the three dimensional model of the VWF-A1 domain; and e) determining which test compound fits into the binding pocket of the three dimensional model of the VWF-A1 protein, thereby identifying which compound would modulate the binding of VWF-A1 protein to GPIb-alpha protein.
 18. A method for identifying a compound that modulates binding of VWF-A1 protein to GPIb-alpha protein, the method comprising: a) providing an electronic library of test compounds; b) providing atomic coordinates listed in Table 8 in a computer readable format for at least 10, 15, 20, 25, 30, 35, or 40 amino acid residues for the A1 domain of the VWF protein, wherein the residues comprise two or more of the following residues: Pro1391, Arg1392, Arg1395, Val1398, Arg1399, Gln1402, Lys1406, Lys1423, Gln1424, Leu1427, Lys1430, or Glu1431; c) converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the VWF-A1 domain; d) performing a data processing method, wherein electronic test compounds from the library are superimposed upon the three dimensional model of the VWF-A1 domain; and e) determining which test compound fits into the binding pocket of the three dimensional model of the VWF-A1 protein, thereby identifying which compound would modulate the binding of VWF-A1 protein to GPIb-alpha protein.
 19. The method of claim 17 or 18, wherein determining comprises detecting an IC₅₀ of less than about 7.5 μg/ml for a test compound.
 20. The method of claim 17 or 18, further comprising: f) obtaining or synthesizing a compound; g) contacting VWF-A1 protein with the compound under a condition suitable for GPIb-alpha-VWF-A1 binding; and h) determining whether the compound modulates GPIb-alpha-VWF-A1 protein binding using a diagnostic assay.
 21. The method of claim 20, wherein contacting comprises perfusing platelets into a flow chamber at a shear flow rate of at least 100 s⁻¹, wherein mutant murine VWF-A1 protein is immobilized on a bottom surface of the chamber.
 22. The method of claim 20, wherein contacting comprises perfusing platelets into the transgenic non-human animal of claim
 4. 23. The method of claim 21, wherein contacting occurs sequentially.
 24. The method of claim 21, wherein the perfusing of platelets occurs prior to administration of the compound.
 25. The method of claim 21, wherein the platelets are human platelets.
 26. The method of claim 21, wherein the platelets are not murine platelets.
 27. The method of claim 20, wherein the determining comprises detecting an increase or decrease in the dissociation rate between VWF-A1 protein and GPIb-alpha protein by at least two-fold.
 28. The method of claim 20, wherein the determining comprises detecting an increase or decrease of platelet adhesion to a surface expressing VWF-A1 protein.
 29. The method of claim 20, wherein the determining comprises detecting an increase or decrease in a stabilization of an interaction between VWF-A1 protein and GPIb-alpha protein.
 30. The method of claim 20, wherein the determining comprises detecting thrombosis formation.
 31. The method of claim 20, wherein the determining comprises identifying an occurrence of an abnormal thrombotic event in the subject.
 32. The method of claim 31, wherein an abnormal thrombotic event comprises abnormal bleeding, abnormal clotting, death, or a combination thereof.
 33. The method of claim 20, wherein the determining comprises dynamic force microscopy, a coagulation factor assay, a platelet adhesion assay, thrombus imaging, a bleeding time assay, aggregometry, review of real-time video of blood flow, a Doppler ultrasound vessel occlusion assay, or a combination thereof.
 34. The method of claim 21, wherein perfusing platelets is followed by perfusion of a labeled agent.
 35. The method of claim 34, wherein the labeled agent comprises one or more of a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, or a small molecule ligand.
 36. The method of claim 34, wherein the agent targets a platelet receptor, a VWF protein, or a portion thereof.
 37. A nucleic acid encoding the protein of any of claims 1-3.
 38. A vector encoding the nucleic acid of claim
 37. 39. An animal expressing the protein of any of claims 1-3.
 40. A method for treating von Willebrand Disease (VWD) in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound that promotes platelet adhesion in the subject, wherein the compound increases the dissociation rate between VWF-A1 protein and GPIb-alpha protein by at least two-fold, thereby administration of the compound increases blood coagulation in the subject.
 41. The method of claim 40, wherein coagulation is measured by a coagulation factor assay, an ex-vivo flow chamber assay, or a combination thereof.
 42. A method for rapidly detecting an internal vascular injury site in a subject, the method comprising: a) administering to a subject a targeted molecular imaging agent, wherein the molecule circulates for an effective period of time in order to bind to the injury site within the subject; b) tracking a deposition of the labeled targeted molecular imaging agent in the subject; and c) identifying the site of a thrombus formation in the subject by imaging the targeted molecular imaging agent, thereby the deposition of the targeted molecular imaging agent at the internal vascular injury site is indicative of internal bleeding within a subject.
 43. The method of claim 42, wherein the targeted molecular imaging agent is administered by subcutaneous, intra-muscular, intra-peritoneal, or intravenous injection; infusion; by oral, nasal, or topical delivery; or a combination thereof.
 44. The method of claim 42, wherein the targeted molecular imaging agent comprises a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, a peptide, a small molecule ligand, or a combination thereof.
 45. The method of claim 44, wherein the nanoparticle comprises a perfluorocarbon.
 46. The method of claim 44, wherein the nanoparticle is coupled to an antibody, a small molecule, a peptide, or a receptor trap.
 47. The method of claim 42, wherein the targeted molecular imaging agent specifically binds to a platelet receptor, or a VWF protein, or a portion thereof.
 48. The method of claim 42, wherein the targeted molecular imaging agent has a T_(1/2) of at least 30 minutes.
 49. The method of claim 42, wherein imaging comprises a PET scan, MRI, IR scan, ultrasound, nuclear imaging, or a combination thereof.
 50. The method of claim 42, wherein the subject is further administered a thrombotic compound.
 51. The method of claim 50, wherein the compound increases the dissociation rate between VWF-A1 protein and GPIb-alpha protein by at least two-fold.
 52. A method for determining whether platelet function or morphology in a subject is abnormal, the method comprising: a) affixing a molecule comprising a murine VWF-A1 domain to a surface of a flow chamber, wherein the domain comprises at least one mutation at a position selected from the group consisting of 1263>S, 1269>D, 1274>R, 1287>R, 1302>D, 1308>R, 1313R>W, 1314>V, 1326>H, 1329>I, 1330>G, 1333>D, 1344>A, 1347>V, 1350>A, 1370>S, 1379>R, 1381>A, 1385>M 1391>Q, 1394>S, 1397>F, 1421>N, 1439>V, 1442>S, 1449>Q, 1466>P, 1469>L, 1472>H, 1473>M, 1475>Q, 1479>G, and any combination thereof, where the position corresponds to an amino acid position of human von Willebrand Factor A1 protein shown in SEQ ID NO: 6; b) perfusing through the flow chamber a volume of blood or plasma from a subject at a shear flow rate of at least about 100 s⁻¹; c) perfusing a targeted molecular imaging agent into the flow chamber; and d) comparing the flow rate of the blood or plasma from the subject as compared to a normal flow rate, so as to determine whether the subject's platelet function or morphology is abnormal.
 53. The method of claim 52, wherein the affixing comprises (i) affixing an antibody which specifically binds VWF-A1 domain, and (ii) perfusing murine mutant VWF-A1 protein in the flow chamber at a shear flow rate of at least 100 s⁻¹.
 54. The method of claim 52, wherein the targeted molecular imaging agent comprises a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, a peptide, a small molecule ligand, or a combination thereof.
 55. The method of claim 52, wherein the targeted molecular imaging agent binds to a platelet receptor, a platelet ligand, or any region of a VWF protein or a portion thereof.
 56. The method of claim 52, wherein the targeted molecular imaging agent comprises horseradish peroxidase (HRP) coupled to an antibody directed at VWF-A1.
 57. The method of claim 52, wherein the comparing comprises a platelet adhesion assay, fluorescence imaging, a chromogenic indicator assay, a microscopy morphology analysis, or any combination thereof.
 58. The method of claim 52, wherein platelets bound to VWF-A1 are less than about 500 cells/mm².
 59. The method of claim 58, wherein the platelets are substantially spherical.
 60. The method of claim 40, 42, or 52, wherein the subject is a human, a canine, a feline, a murine, a porcine, an equine, or a bovine.
 61. The method of claim 52, wherein the VWF molecule is an antibody, a peptide, or a Fab fragment directed to a VWF polypeptide or a portion thereof.
 62. A method for producing mutant von Willebrand Factor A1 protein that specifically binds human platelets, the method comprising: (a) providing an animal expressing a mutant von Willebrand Factor A1 protein, wherein the mutation causes the platelet binding specificity of the animal von Willebrand Factor A1 protein to change to be specific for human platelets; and (b) harvesting the mutant animal von Willebrand Factor A1 so as to produce von Willebrand Factor A1 protein that specifically binds human platelets.
 63. The method of claim 62, wherein the mutant animal von Willebrand Factor A1 protein comprises at least one mutation comprising 1263P>S, 1269N>D, 1274K>R, 1287M>R, 1302G>D, 1308H>R, 1313R>W, 1314I>V, 1326R>H, 1329L>1,1330E>G, 1333A>D, 1344T>A, 1347I>V, 1350T>A, 1370G>S, 1379H>R, 1381T>A, 1385T>M 1391P>Q, 1394A>S, 1397L>F, 1421S>N, 1439L>V, 1442G>S, 1449R>Q, 1466A>P, 1469Q>L, 1472Q>H, 1473V>M, 1475H>Q, 1479S>G, or any combination thereof.
 64. A method for testing efficacy and toxicity of a gene therapy vector, the method comprising: a) introducing a gene therapy vector into the animal of claim 4, allowing sufficient time for expression of the vector; b) perfusing platelets from a subject into the animal under a condition suitable for GPIb-alpha-VWF-A1 protein binding; and c) determining whether or not a thrombotic event occurs in the animal.
 65. The method of claim 64, wherein the vector comprises a nucleic acid encoding a platelet receptor polypeptide, a platelet ligand polypeptide, or a VWF polypeptide, or a portion thereof.
 66. The method of claim 64, wherein the subject is a human, a dog, a cat, a horse, a pig, or a primate.
 67. The method of claim 64, wherein the platelets are not murine platelets.
 68. The method of claim 64, wherein the thrombotic event comprises blood clotting, abnormal bleeding, abnormal clotting, death, or a combination thereof.
 69. The method of claim 64, wherein the determining comprises dynamic force microscopy, a coagulation factor assay, a platelet adhesion assay, thrombus imaging, a bleeding time assay, aggregometry, review of real-time video of blood flow, a Doppler ultrasound vessel occlusion assay, or a combination thereof.
 70. The method of claim 64, wherein perfusing platelets is followed by perfusion of a labeled agent.
 71. The method of claim 70, wherein the labeled agent comprises one or more of a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, or a small molecule ligand.
 72. The method of claim 71, wherein the agent targets a platelet receptor, a VWF protein, or a portion thereof.
 73. A method for calibrating an aggregometry device or a device for measuring clot formation or retraction, the method comprising: a) providing hematologic data obtained from a subject, wherein blood or platelets from the subject is assessed by the device; b) determining whether or not a thrombotic event occurs in the animal of claim 4, wherein the animal is perfused with a sample of blood or platelets from the subject; and c) correlating data obtained from (b) with the data obtained in (a) so as to calibrate the device, wherein a certain data obtained from the device is indicative of the corresponding thrombotic outcome determined in the animal of claim
 4. 74. The method of claim 73, wherein the thrombotic event comprises blood clotting, abnormal bleeding, abnormal clotting, death, or a combination thereof.
 75. The method of claim 22, wherein contacting occurs sequentially.
 76. The method of claim 22, wherein the perfusing of platelets occurs prior to administration of the compound.
 77. The method of claim 22, wherein the platelets are human platelets.
 78. The method of claim 22, wherein the platelets are not murine platelets.
 79. The method of claim 22, wherein perfusing platelets is followed by perfusion of a labeled agent.
 80. The method of claim 79, wherein the labeled agent comprises one or more of a nanoparticle, a fluorophore, a quantum dot, a microcrystal, a radiolabel, a dye, a gold biolabel, an antibody, or a small molecule ligand.
 81. The method of claim 79, wherein the agent targets a platelet receptor, a VWF protein, or a portion thereof. 